TWI796563B - Methods of making antibodies - Google Patents

Abstract

Provided are,inter alia, methods of improving pairing of a heavy chain and a light chain of an antibody (such as a bispecific antibody). Also provided are antibodies (e.g., bispecific antibodies) generated using such methods, libraries, and methods of screening such libraries.

Description

Methods of making antibodies

The development of bispecific antibodies as therapeutic agents for human diseases has great clinical potential. However, the production of bispecific antibodies in the IgG format has been challenging because antibody heavy chains have evolved to associate with antibody light chains in a relatively promiscuous manner. As a result of this promiscuous pairing, the concomitant expression of two antibody heavy chains and two antibody light chains in a single cell naturally results in, for example, heavy chain homodimerization and disorder of heavy chain/light chain pairing.

One approach to circumvent the heavy chain homodimerization problem, termed "knob", aims to facilitate the pairing of two different antibody heavy chains by introducing mutations into the _CH3 domain to modify the contact interface. On one heavy chain, the original amino acid is replaced by an amino acid with a shorter side chain to create a "hole". Conversely, amino acids with large side chains are introduced into other _CH3 domains to create "knobs." By co-expressing these two heavy chains (and two consensus light chains, which must fit into both heavy chains), heterodimer formation ("knob-hole") versus homodimer formation ( "mortar-mortar" or "pestle-pettle") (Ridgway, JB, Protein Eng. 9 (1996) 617-621; Merchant et al. "An efficient route to human bispecific IgG." Nat Biotechnol. 1998; 16 :677-81; Jackman et al. "Development of a two-part strategy to identify a therapeutic human bispecific antibody that inhibits IgE receptor signaling." J Biol Chem. 2010;285:20850-9; and WO 96/027011).

Minimizing heavy chain/light chain scrambling has been made more difficult due to the complex multi-domain heterodimeric interactions within antibody Fabs. Bispecific antibody formats designed to resolve heavy chain/light chain scrambling include: DVD-Ig (Dual Variable Domain Ig) (Nature Biotechnology 25, 1290-1297 (2007)); Interchange Ig (CROSSMAB™) (Schaefer W et al (2011) PNAS 108(27): 11187-11192); 2-in-1 Ig (Science 2009, 323, 1610); BiTE® antibody (PNAS 92(15):7021-7025; 1995); and Lewis et al (2014) "Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface." Nat Biotechnol 32, 191-8; Liu et al. (2015) "A Novel Antibody Engineering Strategy for Making Monovalent Bispecific Heterodimeric IgG Antibodies by Electrostatic Steering Mechanism.” J Biol Chem . Published online January 12, 2015, doi:10.1074/jbc.M114.620260; Mazor et al. 2015. “Improving target cell specificity using a novel monovalent bispecific IgG design.” Mabs. 2015 Published online on January 26, 2015, doi: 10.1080/19420862.2015.1007816; strategies described in WO 2014/081955, WO 2014/082179 and WO 2014/150973.

Nonetheless, there remains a need in the art for methods to reduce mismatched heavy/light chain by-products and increase the yield of correctly assembled bispecific antibodies.

A method for improving the preferential pairing of the heavy and light chains of an antibody is provided, the method comprising the steps of: at least one amino acid at position 94 of the light chain variable domain (V _L ) or at position 96 of the V _L is selected from Uncharged residues are substituted with charged residues selected from the group consisting of aspartic acid (D), arginine (R), glutamic acid (E) and lysine (K), where the amino acid numbering is According to Kabat. In some embodiments, the method comprises the step of substituting each of the amino acids at positions 94 and 96 from an uncharged residue to a charged residue. In some embodiments, the amino acid at position 94 is substituted with D. In some embodiments, the amino acid at position 96 is substituted with R. In some embodiments, the amino acid at position 94 is substituted with D and the amino acid at position 96 is substituted with R. In some embodiments, the amino acid at position 95 of the heavy chain variable domain ( _VH ) is substituted from an uncharged residue to a residue selected from the group consisting of aspartic acid (D), arginine (R), gluten Charged residues of the group consisting of amino acid (E) and lysine (K), where amino acid numbering is according to Kabat. In some embodiments, the amino acid at position 94 of _VL is substituted with D, the amino acid at position 96 of _VL is substituted with R, and the amino acid at position 95 of _VH is substituted with D.

In some embodiments, the methods provided herein further comprise subjecting an antibody (eg, an antibody that has been modified to improve the preferential pairing of heavy and light chains) to at least one affinity maturation step, wherein the substituted amine at position 94 of the _VL Amino acids were not randomized. Additionally or alternatively, in some embodiments, the substituted amino acid at position 96 of the _VL is not randomized. Additionally or alternatively, in some embodiments, the substituted amino acid at position 95 of the _VH is not randomized.

In some embodiments, the antibody is an antibody fragment selected from the group consisting of Fab, Fab', F(ab') ₂ , one-armed antibodies, and scFv or Fv. In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antibody comprises a human IgG Fc region. In some embodiments, the human IgG Fc region is a human IgGl, human IgG2, human IgG3 or human IgG4 Fc region. In some embodiments, the antibody is a monospecific antibody. In some embodiments, the antibodies are multispecific antibodies.

In some embodiments, multispecific antibodies are bispecific antibodies. In some embodiments _, the bispecific antibody comprises a first _CH2 domain ( _CH21 ), _a first _CH3 domain ( _CH31 ), a second _CH2 domain ( _{CH2 2} ) and _a second _CH3 domain; _{wherein CH32} is altered such that within the _CH31 / _CH32 interface _, one or more amino acid residues are One or more amino acid residues are substituted to create a bump on _{the surface of CH32 that interacts with CH31 ; and wherein CH31 is altered} such that _CH31 / _CH Within the 3 ₂ interface, one or more amino acid residues are replaced by amino acid residues with smaller side chain volumes, thereby creating a cavity on the surface of CH _{3 1} that interacts with CH _{3 2} . In some embodiments _, the bispecific antibody comprises a first _CH2 domain ( _CH21 ), _a first _CH3 domain ( _CH31 ), a second _CH2 domain ( _{CH2 2} ) and _a second _CH3 domain; _{wherein CH31} is altered such that within the _CH31 / _CH32 interface, one _or more amino acid residues are modified with a larger side chain volume One or more amino acid residues are substituted so as _to create a bump on _{the surface} of _CH31 that interacts with _CH32 ; and _{wherein CH32} is altered such that _CH31 / _CH Within the 3 ₂ interface, one or more amino acid residues are replaced by amino acid residues with smaller side chain volumes, thereby creating a cavity on the surface of CH _{3 2} that interacts with CH _{3 1} . In some embodiments, the bump is a knob mutation. In some embodiments, the knob mutation comprises T366W, wherein amino acid numbering is according to the EU index. In some embodiments, the pocket is a socket mutation. In some embodiments, the hole mutation comprises at least one, at least two, or all three of T366S, L368A, and Y407V, wherein amino acid numbering is according to the EU index.

Also provided are antibodies produced by any one (or combination) of the methods described herein.

Cross References to Related Applications

This application claims priority to U.S. Provisional Application No. 62/845,59, filed April 9, 2019, the contents of which are hereby incorporated by reference in their entirety. Submit sequence listing on ASCII text file

The following submissions on ASCII text file are hereby incorporated by reference in their entirety: Sequence Listing in Computer Readable Format (CRF) (File Name: 146392047743SEQLIST.TXT, Date of Record: May 4, 2020, Size: 9 KB).

Bispecific antibodies are a promising class of therapeutic agents because their dual specificities allow, for example, delivery of payloads to targeted sites, simultaneous blockade of two signaling pathways, delivery of immune cells to tumor cells, etc. . However, the production of bispecific antibodies (such as bispecific IgG or "BsIgG") remains technically challenging because co-expression of two antibody heavy chains and two antibody light chains in a single cell naturally results in, for example, heavy chain Homodimerization and scrambling of heavy chain/light chain pairing. The methods described herein are based on the applicant's discovery that preferential antibody heavy chain/antibody light chain can be strongly influenced by residues at specific amino acid positions in CDR-H3 and CDR-L3. Furthermore, Applicants found that in many cases the transfer of such residues to the corresponding amino acid positions in other unrelated antibodies increased the yield of correctly assembled BsIgG.

Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2nd ed., John Wiley and Sons, New York (1994) and Hale and Margham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) for those skilled in the art A comprehensive dictionary is provided for many of the terms used in this invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. Numerical ranges include the numerical values defining the range. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amine to carboxy orientation, respectively. Practitioners refer especially to Sambrook et al., 1989 and Ausubel FM et al., 1993 for the definition and terminology of this technique. It is to be understood that this invention is not limited to the particular methodology, protocols and reagents described as these may vary.

Numerical ranges include the numerical values defining the range.

Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amine to carboxy orientation, respectively.

The headings provided herein do not limit the various aspects or embodiments that can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole. definition

The term "antibody" herein is used in the broadest sense and refers to any immunoglobulin (Ig) molecule comprising two heavy chains and two light chains, and any fragment, mutant, variant or derivative thereof, so long as it Exhibiting the desired biological activity (such as epitope binding activity) is sufficient. Examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments as described herein. Antibodies can be mouse, chimeric, human, humanized and/or affinity matured.

As a frame of reference, immunoglobulin as used herein will refer to the structure of immunoglobulin G (IgG). However, those skilled in the art will appreciate/recognize that antibodies of any immunoglobulin class may be utilized in the methods of the invention described herein. For clarity, an IgG molecule contains a pair of heavy chains (HC) and a pair of light chains (LC). Each LC has one variable domain ( _VL ) and one constant domain ( _CL ), while each HC has one variable ( _VH ) and three constant domains ( _CH1 , _CH2 , and _CH 3). The _CH1 and _CH2 domains are connected by a hinge region. Such structures are well known in the art.

Briefly, the basic 4-chain antibody unit is a heterotetrameric glycoprotein consisting of two light (L) chains and two heavy (H) chains (IgM antibodies consist of 5 basic heterotetrameric units together with The J chain consists of additional polypeptides and thus contains 10 antigen-binding sites, whereas secreted IgA antibodies can polymerize to form multivalent assemblies comprising 2-5 basic 4-chain units together with the J chain). In the case of IgG, the 4-chain unit is typically about 150,000 Daltons. Each L chain is linked to an H chain by one covalent disulfide bond, and two H chains are linked to each other by one or more disulfide bonds, depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain ( _VH ) at the N-terminus, followed by three constant domains ( _CH ) of each of the α and γ chains and four _CH domains of the µ and ε isotypes . Each L chain has a variable domain (V _L ) at its N-terminus, followed by a constant domain ( _CL ) at its other end. The VL is aligned with _{the VH and} the _CL is aligned with the first constant domain of the heavy chain ( _CH1 ). Certain amino acid residues are believed to form the interface between the light and heavy chain variable domains. The pairing of _VH and _VL together forms a single antigen binding site. For the structure and properties of different classes of antibodies see, for example, Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton and Lange, Norwalk, CT, 1994, pp. 71 pages and Chapter 6.

L chains from any vertebrate species can be assigned to one of two distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domain of the immunoglobulin heavy chain (Ch), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, γ, ε, and µ, respectively. The gamma and alpha classes are further divided into subclasses based on relatively minor differences in _CH sequence and function, for example, humans exhibit the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.

The term " _CL domain" encompasses the constant region domain of an immunoglobulin light chain, for example extending from about Kabat position 107A to 216 (EU position 108-214 (κ)). The Eu/Kabat conversion table for the kappa C domain is available online from www(dot)imgt(dot)org/IMGTScientificChart/Numbering/Hu_IGKCnber.html and the Eu/Kabat conversion table for the lambda C domain is available online from www(dot)imgt(dot )org/IMGTScientificChart/Numbering/Hu_IGLCnber.html Available online. The _CL domain is adjacent to the _VL domain and includes the carboxy-terminus of the immunoglobulin light chain.

The term " _CH1 domain" of a human IgG as used herein comprises the first (amino-terminal last) constant region domain of an immunoglobulin heavy chain, which extends, for example, from about position 114 to position 223 in the Kabat numbering system (EU position 118-215). The _CH1 domain is adjacent to the _VH domain and is amino-terminal to the hinge region of an immunoglobulin heavy chain molecule, does not form part of the Fc region of an immunoglobulin heavy chain, and is capable of being constant with an immunoglobulin light chain Domains (ie, "CL") dimerize. The EU/Kabat conversion table for the IgGl heavy chain is available online at www(dot)imgt(dot)org/IMGTScientificChart/Numbering/Hu_IGHGnber.html.

The term " _CH2 domain" of a human IgG Fc region generally comprises residues about 231 to about 340 of IgG according to the EU numbering system. The _CH2 domain is unique in that it is not tightly paired with another domain. Instead, two N-linked branched carbohydrate chains are interposed between the two _CH2 domains of the intact native IgG molecule. It has been speculated that carbohydrates may provide an alternative to domain-domain pairing and help stabilize the _CH2 domain. Burton, Mol. lmmunol. 22:161-206 (1985).

The term " _CH3 domain" includes the residues C-terminal to the _CH2 domain in the Fc region (i.e., from about amino acid residue 341 to about amino acid residue 341 of IgG according to the EU numbering system base 447).

The term "Fc region" as used herein generally refers to a dimeric complex comprising the C-terminal polypeptide sequence of an immunoglobulin heavy chain, wherein the C-terminal polypeptide sequence is a polypeptide sequence obtainable by papain digestion of an intact antibody. The Fc region may comprise native or variant Fc sequences. While the boundaries of the Fc sequence of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc sequence comprises about position Cys226, or from about position Pro230 to the carboxy-terminus of the Fc sequence. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU Index, as in Kabat et al., Sequences of Proteins of Immunological Interest , 5th Edition Public Health Service , National Institutes of Health, Bethesda, MD, 1991. The Fc sequence of an immunoglobulin generally comprises two constant domains, a _CH2 domain and a _CH3 domain, and optionally a _CH4 domain. The "Fc polypeptide" herein refers to one of the polypeptides constituting the Fc region, such as monomeric Fc. Fc polypeptides may be obtained from any suitable immunoglobulin, such as human IgGl, IgG2, IgG3 or IgG4 subtypes, IgA, IgE, IgD or IgM. Fc polypeptides can be obtained from mice, such as mouse IgG2a. The Fc region comprises the carboxy-terminal portion of two H chains held together by a disulfide bond. The effector functions of antibodies are determined by sequences in the Fc region; this region is also the part recognized by Fc receptors (FcR) found on certain types of cells. In some embodiments, the Fc polypeptide comprises a portion or all of the wild-type hinge sequence (typically at its N-terminus). In some embodiments, the Fc polypeptide does not comprise a functional or wild-type hinge sequence.

"Fc component" as used herein refers to the hinge region, _CH2 domain or _CH3 domain of the Fc region.

In certain embodiments, the Fc region comprises an IgG Fc region, preferably derived from a wild-type human IgG Fc region. In certain embodiments, the Fc region is derived from "wild type" mouse IgG, such as mouse IgG2a. "Wild-type" human IgG Fc or "wild-type" mouse IgG Fc refers to an amino acid sequence that occurs naturally in the human or mouse population, respectively. Of course, just as the Fc sequence may vary slightly between individuals, one or more changes may be made to the wild-type sequence and still be within the scope of the invention. For example, the Fc region may contain alterations such as mutations in glycosylation sites or include unnatural amino acids.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in antibody binding to antigen. The variable domains of the heavy and light chains ( _VH and _VL , respectively) of primary antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See eg Kindt et al. Kuby Immunology , 61st Ed., WH Freeman and Co., p. 91 (2007).) A single _VH or _VL domain may be sufficient to confer antigen binding specificity. Furthermore, antibodies that bind a particular antigen can be isolated using the _VH or _VL domains, respectively, from antibodies that bind that antigen to screen libraries of complementary _VL or _VH domains. See, eg, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term "hypervariable region" or "HVR" as used herein refers to each of the regions of antibody variable domains that are highly variable in sequence and that determine antigen-binding specificity, such as "complementarity determining regions" ("CDRs") ).

In general, antibodies contain six CDRs: three in the _VH (CDR-H1, CDR-H2, CDR-H3), and three in the _VL (CDR-L1, CDR-L2, CDR-L3) . Exemplary CDRs herein include: (a) present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 ( H2) and hypervariable loops at 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) present at amino acid residues 24-34 (L1 ), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2) and 95-102 (H3) at the CDRs (Kabat et al., Sequences of Proteins of Immunological Interest , 5th edition Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 ( Antigen contacts at L3), 30-35b (H1), 47-58 (H2) and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)).

Unless otherwise indicated, CDRs were determined according to Kabat et al. (supra). Those skilled in the art will appreciate that CDR designations can also be determined according to Chothia (supra), McCallum (supra), or any other scientifically accepted nomenclature system.

"Framework" or "FR" refers to the variable domain residues other than the complementarity determining regions (CDRs). The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3 and FR4. Therefore, CDR and FR sequences generally appear in the following order in V _H (or V _L ): FR1-CDR-H1 (CDR-L1)-FR2-CDR-H2 (CDR-L2)-FR3-CDR-H3 (CDR -L3)-FR4.

The phrases "antigen binding arm", "target molecule binding arm", "target binding arm" and variations thereof as used herein refer to an antibody (such as a bispecific antibody) that has the ability to specifically bind a target of interest. ) components. Typically and preferably, the antigen binding arm is a complex of immunoglobulin polypeptide sequences, such as the CDRs and/or variable domain sequences of immunoglobulin light and heavy chains.

"Target" or "target molecule" refers to the moiety recognized by the binding arm of an antibody, such as a bispecific antibody. For example, if the antibody is a multispecific antibody (eg, a bispecific antibody), the targets may be epitopes on a single molecule or on different molecules, or pathogens or tumor cells, as the case may be. Those skilled in the art will appreciate that a target is determined by the binding specificity of a target binding arm and that different target binding arms may recognize different targets. The target preferably binds to the antibody (eg, a bispecific antibody) with an affinity (according to methods known in the art, including those described herein) of greater than 1 μM Kd. Examples of target molecules include, but are not limited to, serum soluble proteins and/or their receptors (such as cytokines and/or cytokine receptors), adhesins, growth factors and/or their receptors, hormones, virions (such as RSV F protein, CMV, Staphylococcus A, Influenza, Hepatitis C virus), microorganisms (e.g. bacterial cell proteins, fungal cells), adhesins, CD proteins and their receptors.

The term "interface" as used herein refers to the association surface created by the interaction of one or more amino acids in a first antibody domain with one or more amino acids in a second antibody domain. Exemplary interfaces include, for example, _CH1 / _CL , _VH / _VL , and _CH3 / _CH3 . In some embodiments, the interface includes, for example, hydrogen bonds, electrostatic interactions, or salt bridges between amino acids forming the interface.

One example of a "intact" or "full length _" antibody is an antibody comprising an antigen binding arm as well as _CL and at least the heavy chain constant domains CHI, _CH2 and _CH3 . The constant domain can be a native sequence constant domain (eg, a human native sequence constant domain) or an amino acid sequence variant thereof.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical and/or bind the same epitope, except, for example, containing naturally occurring Mutations or possible variant antibodies that appear during the production of monoclonal antibody preparations, such variants generally exist in trace amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), monoclonal antibody preparations have each monoclonal antibody directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method. For example, monoclonal antibodies according to the invention can be produced by a variety of techniques including, but not limited to, the fusionoma method, recombinant DNA methods, phage display methods and the use of transgenic animals containing all or a portion of the human immunoglobulin loci Methods for making monoclonal antibodies, such methods and other exemplary methods for making monoclonal antibodies are described herein.

"Naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or radiolabeled. Naked antibodies can be present in pharmaceutical compositions.

"Primary antibody" refers to a naturally occurring immunoglobulin molecule of varying structure. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons consisting of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable domain ( _VH ), also known as a variable heavy domain or heavy chain variable region, followed by three constant heavy domains ( _CH1 , C _H2 and _CH3 ). Similarly, from N-terminus to C-terminus, each light chain has a variable domain ( _VL ), also known as a variable light domain or light chain variable region, followed by a constant light ( _CL ) domain.

"Monospecificity" refers to the ability of an antibody to bind only one epitope. "Bispecific" refers to the ability of an antibody to bind two different epitopes. "Multispecificity" refers to the ability of an antibody to bind more than one epitope. In certain embodiments, multispecific antibodies encompass bispecific antibodies. For bispecific and multispecific antibodies, the epitopes can be on the same antigen, or each epitope can be on a different antigen. In certain embodiments, bispecific antibodies bind to two different antigens. In certain embodiments, bispecific antibodies bind to two different epitopes on one antigen. In certain embodiments, the multispecific antibody (such as a bispecific antibody) is present at about ≤ 1 μM, about ≤ 100 nM, about ≤ 10 nM, about ≤ 1 nM, about ≤ 0.1 nM, about ≤ 0.01 nM, or about A dissociation constant (Kd) of ≤ 0.001 nM (eg, about 10 ^-8 M or less, eg, about 10 ^-8 M to about 10 ^-13 M, eg, about 10 ^-9 M to about 10 ^-13 M) binds to each antigen Determine base.

The term "multispecific antibody" is used herein in the broadest sense to refer to an antibody capable of binding two or more antigens. In certain aspects, a multispecific antibody refers to a bispecific antibody, such as a human bispecific antibody, a humanized bispecific antibody, a chimeric bispecific antibody, or a mouse bispecific antibody.

An "antibody fragment" comprises a portion of an intact antibody, preferably the _VH and _VL of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, ScFv and Fv fragments; single-armed antibodies and multispecific antibodies formed from antibody fragments.

Antibodies may be "chimeric" antibodies in which part of the heavy and/or light chain(s) is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular class or subclass of antibodies, and part of the chain(s) The remainder is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, provided that such fragments exhibit the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies contemplated herein include primatized antibodies comprising variable domain antigen binding sequences derived from a non-human primate (eg, Old World monkey, ape, etc.) and human constant region sequences.

"Humanized" forms of non-human (eg, rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. In most cases, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the hypervariable region of the recipient are replaced by those from a species such as mouse, rat, rabbit or non-human primate. Substitution of residues in the hypervariable regions of a non-human species (donor antibody), the humanized antibodies possess the desired antibody specificity, affinity and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody potency. In general, a humanized antibody will comprise substantially all of at least one and typically two variable domains in which all or substantially all hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of them All FRs are those of human immunoglobulin sequences. A humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 ( 1992).

The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation which is in a form which permits the effective biological activity of the active ingredients contained therein and which is free from unacceptable toxicity to the individual to whom the pharmaceutical composition is to be administered. additional components.

"Pharmaceutically acceptable carrier" means an ingredient of a pharmaceutical composition or formulation other than the active ingredient, which is nontoxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

As used herein, "complex" or "complex" refers to two or more compounds that interact with each other via bonds and/or forces (e.g., van der Waals force, hydrophobic force, hydrophilic force) that are not peptide bonds. Association of more molecules. In one embodiment, the complex is heteromultimeric. It will be appreciated that the term "protein complex" or "polypeptide complex" as used herein includes non-proteinaceous entities (including, but not limited to, chemical molecules such as toxins or detectors, for example) that have proteins incorporated into the protein complex. Complex.

An antibody that "binds an antigen of interest" (such as a monospecific or multispecific antibody) is an antibody that binds an antigen, such as a protein, with sufficient affinity such that the antibody is useful for targeting the protein or a cell or tissue expressing the protein Diagnostic and/or therapeutic agents, and do not significantly interact with other proteins. In such embodiments, the antibody will bind to the "non-target" protein to a lesser extent than the antibody to its specific target, as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA) or ELISA Approximately 10% of protein binding. With respect to the binding of an antibody to a target molecule, the term "specifically binds" or "specifically binds to" or "specifically for" a particular polypeptide or an epitope on a particular polypeptide target means measurably different A specific interaction (eg a non-specific interaction may be binding to bovine serum albumin or casein). Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule. For example, specific binding can be determined by competition with a control molecule similar to the target, such as an excess of a non-labeled target. In such cases, specific binding is indicated if binding of the labeled target to the probe is competitively inhibited by excess unlabeled target. As used herein, the term "specifically binds" or "specifically binds to" or "specifically for" a particular polypeptide or an epitope on a particular polypeptide target may, for example, be exhibited by a molecule having a Kd for the target of at least About 200 nM, or at least about 150 nM, or at least about 100 nM, or at least about 60 nM, or at least about 50 nM, or at least about 40 nM, or at least about 30 nM, or at least about 20 nM, or at least about 10 nM, or at least about 8 nM, or at least about 6 nM, or at least about 4 nM, or at least about 2 nM, or at least about 1 nM, or higher affinity. In one embodiment, the term "specifically binds" refers to the binding of a multispecific antibody to a particular polypeptide or an epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

"Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (eg, an antibody, such as a bispecific or multispecific antibody) and its binding partner (eg, an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (Kd). For example, the K can be about 200 nM or less, about 150 nM or less, about 100 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, about 10 nM or less, about 8 nM or less, about 6 nM or less, about 4 nM or less, about 2 nM or less, or about 1 nM or less. Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high affinity antibodies generally bind antigen faster and tend to remain bound longer. Various methods of measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention.

In one embodiment, "Kd" or "Kd value" is measured by using a surface plasmon resonance assay. For example, Kd values can be measured in -10 response units ( RU) to determine. Briefly, in one example, N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxybutanedi Carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) were activated with imide (NHS). Antigen was diluted to 5 μg/ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 μl/min to achieve approximately 10 response units (RU) of coupled protein. After antigen injection, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (eg, 0.78 nM to 500 nM) were injected in PBS containing 0.05% Tween 20 (PBST) at 25°C at a flow rate of approximately 25 μl/min. Association rates (k _on ) and dissociation rates were calculated by simultaneously fitting association and dissociation sensorgrams using a simple 1:1 Langmuir binding model (BIAcore Evaluation Software version 3.2) (k _off ). Equilibrium dissociation constants (Kd) were calculated as the ratio k _off /k _on . See, eg, Chen et al., J. Mol. Biol. 293:865-881 (1999). If the association rate exceeds 10 ⁶ M ⁻¹ s ⁻¹ by the surface plasmon resonance assay described above, the association rate can be determined by using the fluorescence quenching technique measured at 25 Increase in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab format) in PBS (pH 7.2) in the presence of increasing concentrations of antigen at °C Or decrease, as measured in a spectrometer, such as a spectrophotometer equipped with a stopped flow (Aviv Instruments) or an 8000 series SLM-Aminco spectrophotometer with a stirred optical cell (ThermoSpectronic).

Unless otherwise specified, references to antibodies (e.g., modified antibodies, such as modified bispecific antibodies), such as antibodies (e.g., bispecific antibodies), fragments or derivatives thereof, produced according to the methods provided herein are " "Biologically active" and "bioactive" and "biocharacteristic" mean having the ability to bind to a biological molecule.

"Isolated" when used to describe various heteromultimeric polypeptides means a heteromultimer that has been separated and/or recovered from the cell or cell culture in which it is expressed. Pollutant components of their natural environment are substances that would interfere with the diagnostic or therapeutic use of the heteromultimer, and may include enzymes, hormones and other proteinaceous or nonproteinaceous solutes. In certain embodiments, the heteromultimer will be purified (1) to greater than 95% by weight protein as determined by the Lowry method, and optimally to greater than 99% by weight; (2) to a level sufficient to obtain The extent of the N-terminal or internal amino acid sequence of at least 15 residues determined by using a spin cup sequencer; or (3) achieved by SDS PAGE under reducing or non-reducing conditions using Coomassie blue (Coomassie blue) or preferably silver staining to determine homogeneity. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.

Antibodies, such as bispecific antibodies, are generally purified to substantial homogeneity. The phrases "substantially homogeneous", "substantially homogeneous form" and "substantially homogeneous" are used to indicate that the product is substantially free from combinations of undesired polypeptides such as heavy chain homodimers and/or scrambled heavy chain/light chain pair).

Expressed in terms of purity, substantial homogeneity means that the amount of by-products does not exceed 10%, 9%, 8%, 7%, 6%, 4%, 3%, 2%, or 1% by weight , or less than 1% by weight. In one embodiment, by-products are less than 5%.

"Biomolecules" means nucleic acids, proteins, carbohydrates, lipids, and combinations thereof. In one embodiment, the biomolecule occurs in nature.

Unless the context indicates otherwise, the terms "first" polypeptide (such as a heavy chain (HC1 or HC ₁ ) or light chain (LC1 or LC ₁ )) and "second" polypeptide (such as a heavy chain (HC2 or HC ₂ ) or Light chain (LC2 or LC ₂ )) and variants thereof are generic designations only and should not be considered to identify a specific or particular polypeptide or component of an antibody (such as a bispecific antibody) produced using the methods provided herein.

Unless otherwise indicated, commercially available reagents mentioned in the examples were used according to the manufacturer's instructions. The source of these cells, identified by ATCC deposit number in the examples below and throughout this specification, was the American Type Culture Collection (Manassas, VA). Unless otherwise noted, the present invention employs standard procedures of recombinant DNA technology, such as those described above and in the following textbooks: Sambrook et al. (supra); Ausubel et al., Current Protocols in Molecular Biology (Green Publishing Associates and Wiley Interscience, NY, 1989); Innis et al, PCR Protocols: A Guide to Methods and Applications (Academic Press, Inc., NY, 1990); Harlow et al, Antibodies: A Laboratory Manual (Cold Spring Harbor Press, Cold Spring Harbor, 1988); Gait, Oligonucleotide Synthesis (IRL Press, Oxford, 1984); Freshney, Animal Cell Culture, 1987; Coligan et al., Current Protocols in Immunology, 1991.

Reference herein to "about" a value or parameter refers to the usual error range for the respective value that is readily understood by those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) aspects of that value or parameter itself. For example, description referring to "about X" includes description of "X".

It should be understood that aspects and embodiments of the present invention described herein include "comprising aspects and embodiments", "consisting of aspects and embodiments" and "consisting essentially of aspects and embodiments".

All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety. Method for improving heavy chain / light chain pairing selectivity

The present application is based on the identification of amino acid positions in _VL (e.g., the _VL of an antibody light chain or fragment thereof) and _VH (e.g., the _VH of an antibody heavy chain or fragment thereof) in a preferential heavy chain/light chain pairing Residues that play a role in .

As described in further detail below, the methods provided herein involve introducing one or more substitutions within the variable domains of heavy and/or light chain polypeptides, eg, at particular residues within the CDR sequences. As will be appreciated by those of ordinary skill in the art, various numbering conventions may be employed for designating particular amino acid residues within an antibody variable region sequence. Commonly used numbering conventions include Kabat and EU Index numbering (see Kabat et al., Sequences of Proteins of Immunological Interest , 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Other protocols that include revised or alternative numbering systems for variable domains include Chothia (Chothia C, Lesk AM (1987), J Mal Biol 196: 901-917; Chothia et al. (1989), Nature 342: 877-883 ), IMGT (Lefranc et al. (2003), Dev Comp Immunol 27: 55-77), and AHo (Honegger A, Plückthun A (2001) J Mol Biol 309: 657-670). These references provide the amino acid sequence numbering scheme for the variable region of an immunoglobulin used to define the positions of the variable region amino acid residues of antibody sequences.

Unless expressly stated otherwise herein, all references to amino acid residues (i.e., numbering) of the variable region of an immunoglobulin heavy chain (i.e., _VH ) appearing in the Examples and in the claims are based on Kabat The numbering system, unless specifically indicated otherwise, is the same for all references to _VL residues. All references to immunoglobulin heavy chain constant region _CH1 , _CH2 and _CH3 residues (i.e., numbering) appearing in the Examples and Claims are based on the EU system unless specifically indicated otherwise, Otherwise the same is true for all references to _CL residues. Using knowledge of residue numbering according to Kabat or EU index numbering, one of ordinary skill in the art can identify the amino acid sequence modifications described herein according to any commonly used numbering convention.

Although an item, component or element provided herein (such as "antibody," "substitution" or "substitution mutation") may be described or claimed in the singular, the plural is encompassed herein unless limitation to the singular is expressly stated.

As described in more detail below, provided herein are methods of improving correct heavy chain/light chain pairing in antibodies, including bispecific antibodies, comprising introducing one or more substitutions into the _VH and/or _VL . Also provided are methods of improving the yield of antibodies, such as properly assembled bispecific antibodies, comprising introducing one or more substitutions into the _VH and/or _VL of the antibody, using specific methods such as this technique Methods known in the art) produce antibodies comprising substitutions (eg, bispecific antibodies) in higher yields than unsubstituted antibodies (eg, bispecific antibodies) produced using the same method. Previous efforts have focused on introducing one or more amino acid substitutions into the framework regions of variable domains. See eg Froning et al., Protein Science, 2017, 26:2021-38. Liu et al., J. Biol. Chem. 2015, 290:7535-62. Lewis et al., Nature Biotechnology, 2014, 32:191-202.

In some embodiments, the methods provided herein further comprise introducing one or more modifications in the Fc region to facilitate heterodimerization of the two heavy chains of an antibody, such as a bispecific antibody. Substitution mutations in the heavy and light chain variable domains

Provided herein is a method of improving the pairing (such as preferential pairing) of the heavy and light chains of an antibody, the method comprising the steps of aligning at least one of the light chain variable domains (V _L ) at position 94 or at position 96 of the V _L Substitution of an amino acid (e.g. "original amino acid") from an uncharged residue selected from aspartic acid (D), arginine (R), glutamic acid (E) and lysine (K) The charged residues, where amino acid numbering is according to Kabat. In some embodiments, the method comprises the step of substituting the two amino acids at positions 94 and 96 (e.g., the original amino acids) from uncharged residues to charged residues, such as D, R, E or K. In some embodiments, the method comprises providing an antibody into which one or more of the substitutions discussed above have been introduced. In some embodiments, the method comprises providing an antibody (such as a bispecific or multispecific antibody) that binds one (or more) of the exemplary targets described elsewhere herein.

Preferential pairing describes when a first polypeptide (such as heavy chain) with a second polypeptide (such as a light chain) pairing pattern. In some embodiments, when HC ₁ is co-expressed with at least LC ₁ and LC ₂ , if the amount of HC ₁ /LC ₁ heavy chain-light chain pairing is greater than the amount of HC ₁ /LC ₂ pairing, e.g., in an antibody (e.g. Preferential pairing occurs between HC ₁ and LC ₁ of a bispecific antibody). Likewise, when HC ₂ is co-expressed with at least LC ₁ and LC ₂ , if the amount of HC ₂ /LC ₂ heavy chain-light chain pairing is greater than the amount of HC ₂ /LC ₁ pairing, e.g. in a multispecific antibody (e.g. Preferential pairing occurs between HC ₂ and LC ₂ of a bispecific antibody). As described in further detail elsewhere herein, HC ₁ /LC ₁ , HC ₁ /LC ₂ , HC ₂ /LC ₁ , and HC ₂ /LC ₂ pairings can be determined by methods known in the art, such as liquid chromatography mass spectrometry ( LCMS) measurement.

In some embodiments, the term "original amino acid" refers to the presence at a specific position, such as position 94 of _VL , and/or Amino acid at position 96. In some embodiments, the term "uncharged amino acid" or "uncharged residue" refers to a pH value at physiological pH, such as between about 6.8 and about 7.5, between about 6.9 and about 7.355, or between about 6.95 and about 7.355. An amino acid that is neither positively charged (such as protonated) nor negatively charged (such as deprotonated) at a pH between 7.45. In some embodiments, "charged amino acid" refers to a positively charged amino acid at physiological pH, for example, a pH between about 6.8 and about 7.5, between about 6.9 and about 7.355, or between about 6.95 and 7.45. Amino acids that are charged (such as protonated) or negatively charged (such as deprotonated). In some embodiments, the uncharged amino acid residue is an amino acid residue other than D, R, E, or K. In some embodiments, the amino acid at position 94 (eg, the original amino acid) is substituted with D. In some embodiments, the amino acid at position 96 (eg, the original amino acid) is substituted with R. In some embodiments, the amino acid at position 94 (eg, the original amino acid) is substituted with D, and the amino acid at position 96 (eg, the original amino acid) is substituted with R.

In some embodiments, the method further comprises substituting the amino acid at position 95 (e.g., the original amino acid) of the heavy chain variable domain ( _VH ) from an uncharged residue to a group selected from aspartic acid ( D), Charged residues of arginine (R), glutamic acid (E) and lysine (K), where amino acid numbering is according to Kabat. In some embodiments, the amino acid at position 95 (eg, the original amino acid) is substituted with D. In some embodiments, the amino acid at position 94 of _VL (eg, the original amino acid) is substituted with D, the amino acid at position 96 of _VL (eg, the original amino acid) is substituted with R, and V The amino acid at position 95 of _H (eg, the original amino acid) is substituted with D.

Also provided is a method of improving the pairing of the heavy and light chains of an antibody, such as homologous pairing, i.e., preferential pairing of homologous _VH to VL, _Fab , and HC to LC, comprising the steps of: The amino acid at position 95 of the chain variable domain ( _VH ) (eg, the original amino acid) is substituted from an uncharged residue to a group selected from aspartic acid (D), arginine (R), glutamine Charged residues of acid (E) and lysine (K), where amino acid numbering is according to Kabat. In some embodiments, the amino acid at position 95 (eg, the original amino acid) is substituted with D.

Also provided herein is a method of improving the pairing (such as homologous pairing) of the heavy and light chains of an antibody, the method comprising the steps of aligning position 91 of the light chain variable domain ( _VL ), position 94 of the _VL , or At least one amino acid at position 96 of _VL (e.g. "original amino acid") is substituted from a non-aromatic residue selected from tryptophan (W), phenylalanine (F) and tyrosine (Y) Aromatic residues where amino acid numbering is according to Kabat. In some embodiments, the method comprises the step of substituting at least two amino acids (eg, original amino acids) at position 91, position 94, or position 96 from non-aromatic residues with a group selected from W, F, and Aromatic residue of Y. In some embodiments, the method comprises the step of substituting the amino acids at positions 94 and 96 (e.g., the original amino acids) from non-aromatic residues with aromatic residues selected from W, F, and Y . In some embodiments, the method comprises the step of substituting each of the amino acids at positions 91, 94, and 96 (e.g., the original amino acid) from a non-aromatic residue to a group selected from W, Aromatic residues of F and Y. In some embodiments, the method comprises providing an antibody into which one or more of the substitutions discussed above have been introduced. In some embodiments, the method comprises providing an antibody (such as a bispecific or multispecific antibody) that binds one (or more) of the exemplary targets described elsewhere herein.

In some embodiments, "original amino acid" refers to an amino acid present at position 91, position 94, and/or position 96 of _VL immediately prior to substitution with an aromatic amino acid (e.g., W, F, and Y). Amino acids (eg, non-aromatic amino acids). In some embodiments, the term "non-aromatic amino acid" or "non-aromatic residue" refers to an amino acid that does not contain an aromatic ring. In some embodiments, "non-aromatic residue" refers to an amino acid residue that is not W, F, or Y.

In some embodiments, the amino acid at position 91 (eg, the original amino acid) is substituted with Y. In some embodiments, the amino acid at position 94 (eg, the original amino acid) is substituted with Y. In some embodiments, the amino acid at position 96 (eg, the original amino acid) is substituted with W. In some embodiments, the amino acid at position 91 (eg, the original amino acid) is substituted with Y, and the amino acid at position 94 (eg, the original amino acid) is substituted with Y. In some embodiments, the amino acid at position 91 (eg, the original amino acid) is substituted with Y, and the amino acid at position 96 (eg, the original amino acid) is substituted with W. In some embodiments, the amino acid at position 94 (eg, the original amino acid) is substituted with Y, and the amino acid at position 96 (eg, the original amino acid) is substituted with W. In some embodiments, the amino acid at position 91 (eg, the original amino acid) is substituted with Y, the amino acid at position 94 (eg, the original amino acid) is substituted with Y, and the amino acid at position 96 (eg original amino acid) is substituted with W.

In some embodiments, the method further comprises substituting the amino acid at position 95 (e.g., the original amino acid) of the heavy chain variable domain ( _VH ) from an uncharged residue to a group selected from aspartic acid ( D), Charged residues of arginine (R), glutamic acid (E) and lysine (K), where amino acid numbering is according to Kabat. In some embodiments, the method further comprises substituting the amino acid at position 95 (e.g., the original amino acid) of the heavy chain variable domain ( _VH ) from a non-aromatic residue to one selected from tryptophan ( W), aromatic residues of phenylalanine (F) and tyrosine (Y).

In some embodiments, one or more of the substitutions described above are introduced into an antibody fragment, eg, an antibody fragment comprising a _VL domain and a _VH domain. Such antibody fragments include, but are not limited to, eg, Fab, Fab', monospecific F(ab') ₂ , bispecific F(ab') ₂ , one-armed antibodies, ScFv, Fv, and the like.

In some embodiments, the antibody into which one or more of the substitutions described above is introduced is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antibody comprises a kappa light chain. In some embodiments, the antibody comprises a lambda light chain. In certain embodiments, the _VL comprises framework sequences of a KV1 or KV4 human germline family. In some embodiments, the _VH comprises framework sequences of an HV2 or HV3 human germline family. In some embodiments, the antibody comprises a murine Fc region. In some embodiments, the antibody comprises a human Fc region, such as a human IgG Fc region, eg, a human IgGl, human IgG2, human IgG3m or human IgG4 Fc region. In some embodiments, the antibody is a monospecific antibody. In some embodiments, the antibody is a multispecific antibody, such as a bispecific antibody.

_In certain embodiments, the antibody into which one or more of the substitutions described above is introduced is a bispecific antibody comprising a first V L (V _L 1) paired with a first V _H (V _L 1) And the second V _L (V _L 2) paired with the second V _H (V _H 2), wherein V _L 1 contains the Q38K substitution mutation, V _H 1 contains the Q39E substitution mutation, V _L 2 contains the Q38E substitution mutation, V _H 2 contains the Q39K substitution mutation, where the amino acid numbering is according to Kabat. In some embodiments, _VL1 comprises a Q38E substitution mutation, _VH1 comprises a Q39K substitution mutation, _VL2 comprises a Q38K substitution mutation, _VH2 comprises a Q39E substitution mutation, wherein the amino acid numbering is according to Kabat. It will be apparent to those of ordinary skill in the art that the terms " _VL1 ", " _VH1 ", " _VL2 " and " _VH2 " are arbitrary designations, and for example in any of the examples herein "V _L 1" and "V _L 2" can be reversed.

Additionally or alternatively, in some embodiments, the antibody into which one or more substitutions described above are introduced is _a bispecific antibody comprising: a second _CH1 domain comprising a first CH1 domain ( _CH11 ) A heavy chain (HC ₁ ), a first light chain (LC ₁ ) comprising a first _CL domain ( _CL1 ), a second heavy chain (HC 1 ) comprising a second CH ₁ domain (CH _{1 2} ) ₂ ) and the second light chain (LC ₂ ) comprising the first _CL domain ( _CL2 ). It will be apparent to those of ordinary skill in the art that the terms "HC ₁ ", "HC ₂ ", "LC ₁ ", "LC ₂ ", etc. are arbitrary designations, and for example "HC" in any of the examples herein ₁ ” and “HC ₂ ” can be reversed. That is, any of the mutations described above as being in the _CHI domain of H1 and the _CL domain of L1 may alternatively be in the _CHI domain of H2 and the _CL domain of L2. In some embodiments, the method further comprises substituting S183 in _CH11 with E, _V133 in _CL1 with K, S183 in _CH12 with K, and _V133 in C _L2 with E , where amino acid numbering is according to the EU index. In some embodiments, the method further comprises replacing S183 in _CH11 with _K , _V133 in _CL1 with E, S183 in _CH12 with E and V133 in _CL2 with K , where amino acid numbering is according to the EU index. See eg Dillon et al. (2017) MABS 9(2): 213-230 and WO2016/172485. In some embodiments, HC ₁ further comprises a first _CH 2 ( _CH 2 ₁ ) domain and/or a first _CH 3 ( _CH 3 ₁ ) domain. Additionally or alternatively, in some embodiments, HC ₂ further comprises a second _CH 2 ( _CH 2 ₂ ) domain and/or a second _CH 3 ( _CH 3 ₂ ) domain. In some embodiments, _CH32 is modified such that within _{the CH31} / _CH32 interface, one _or more amino acid residues are replaced by one or more amine groups with larger side chain volumes _. Acid residues are replaced, thereby creating a bump on the surface of _CH32 that _interacts with _{CH31 ;} and _CH31 is altered such that within the _CH31 / _CH32 interface _{, one or} Multiple amino acid residues are _replaced by amino acid residues with smaller side chain volumes, _thereby creating pockets on the surface of _CH31 that interact with _CH32 . In some embodiments, _CH31 is modified such that within the _CH31 / _CH32 interface, one _or more amino acid residues are replaced by one or more amine groups with larger side chain _{volumes .} Acid residues are replaced, thereby creating _{a bump on the surface of CH31 that interacts with CH32 ; and CH32 is altered so that} within the _CH31 / _CH32 interface, one or Multiple amino acid residues are _replaced by amino acid residues with _smaller side chain volumes, thereby creating pockets on the surface of _CH32 that interact with _CH31 . In some embodiments, the bump is a knob mutation, such as a knob mutation comprising T366W, wherein amino acid numbering is according to the EU index. In some embodiments, the cavity is a hole mutation, eg, a hole mutation comprising at least one, at least two, or all three of T366S, L368A, and Y407V, wherein amino acid numbering is according to the EU index. Additional details regarding knob and hole mutations are provided, for example, in US 5,731,168, US 5,807,706, US 7,183,076, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the HC ₁ /LC ₁ pair of the bispecific antibody binds to a first antigen and the HC ₂ /LC ₂ pair of the bispecific antibody binds to a second antigen. In some embodiments, the HC ₁ /LC ₁ pair of the bispecific antibody binds to a first epitope of the first antigen, and the HC ₂ /LC ₂ pair of the bispecific antibody binds to a second antigen of the first antigen Determine base.

A method of making (such as modifying or engineering) an antibody (such as a bispecific antibody) to obtain a modified antibody (such as a modified bispecific antibody) with improved preferential heavy chain/light chain pairing is provided, the method comprising substituting the amino acid at position 94 of the light chain variable domain ( _VL ) and/or at position 96 of the _VL (e.g. the original amino acid) from an uncharged residue to a group selected from aspartic acid (D ), arginine (R), glutamic acid (E) and lysine (K) charged residues to obtain modified antibodies (such as modified bispecific antibodies), wherein the amino acid numbering is According to Kabat. In some embodiments, the method comprises the step of substituting at least two amino acids (e.g., original amino acids) at positions 94 and 96 from uncharged residues to charged residues, e.g., D, R, E or K to obtain modified antibodies (eg bispecific antibodies). In some embodiments, modified antibodies (eg, bispecific or multispecific antibodies) bind to exemplary targets described elsewhere herein. In many cases, the sequences of the heavy and light chains of antibodies that bind to such targets are publicly available and can be aligned and mapped to the Kabat numbering scheme, followed by scanning against the Kabat sequence database to identify positions to be substituted.

In some embodiments, the amino acid at position 94 (eg, the original amino acid) is substituted with D to obtain a modified antibody (eg, a modified bispecific antibody). In some embodiments, the amino acid at position 96 (eg, the original amino acid) is substituted with R to obtain a modified antibody (eg, a modified bispecific antibody). In some embodiments, the amino acid at position 94 (eg, the original amino acid) is substituted with D, and the amino acid at position 96 (eg, the original amino acid) is substituted with R to obtain a modified antibody (eg, Modified bispecific antibody).

In some embodiments, the method further comprises substituting the amino acid at position 95 (e.g., the original amino acid) of the heavy chain variable domain ( _VH ) from an uncharged residue to a group selected from aspartic acid ( D), charged residues of arginine (R), glutamic acid (E) and lysine (K) to obtain modified antibodies (such as modified bispecific antibodies), wherein the amino acid numbers Department according to Kabat. In some embodiments, the amino acid at position 95 (eg, the original amino acid) is substituted with D to obtain a modified antibody (eg, a modified bispecific antibody). In some embodiments, the amino acid at position 94 of _VL (eg, the original amino acid) is substituted with D, the amino acid at position 96 of _VL (eg, the original amino acid) is substituted with R, and V The amino acid at position 95 of _H (eg, the original amino acid) is substituted with D to obtain a modified antibody (eg, a modified bispecific antibody).

Also provided is a method of making (such as modifying or engineering) an antibody (such as a bispecific antibody) to obtain a modified antibody (such as a modified bispecific antibody) with an improved preferential heavy chain/light chain pairing, the The method involves substituting the amino acid at position 95 (e.g., the original amino acid) of the heavy chain variable domain ( _VH ) from an uncharged residue to a residue selected from aspartic acid (D), arginine (R ), glutamic acid (E) and lysine (K) to obtain modified antibodies (eg modified bispecific antibodies), wherein the amino acid numbering is according to Kabat. In some embodiments, the amino acid at position 95 (eg, the original amino acid) is substituted with D to obtain a modified antibody (eg, a modified bispecific antibody).

Also provided is a method of making (such as modifying or engineering) an antibody (such as a bispecific antibody) to obtain a modified antibody (such as a modified bispecific antibody) with an improved preferential heavy chain/light chain pairing, the The method comprises substituting the amino acid at position 91 of the light chain variable domain (V _L ), at position 94 of the V _L and/or at position 96 of the V _L (eg, the original amino acid) from a non-aromatic residue to Aromatic residues selected from tryptophan (W), phenylalanine (F) and tyrosine (Y) to obtain modified antibodies (eg modified bispecific antibodies), wherein the amino acid numbering is according to Kabat. In some embodiments, the method comprises the step of substituting at least two amino acids (eg, original amino acids) at position 91, position 94, or position 96 from non-aromatic residues with a group selected from W, F, and Aromatic residues of Y to obtain modified antibodies (eg modified bispecific antibodies). In some embodiments, the method comprises the step of substituting the amino acids at positions 94 and 96 (e.g., the original amino acids) from non-aromatic residues with aromatic residues selected from W, F, and Y To obtain modified antibodies (eg modified bispecific antibodies). In some embodiments, the method comprises the step of substituting each of the amino acids at positions 91, 94, and 96 (e.g., the original amino acid) from a non-aromatic residue to a group selected from W, Aromatic residues of F and Y to obtain modified antibodies (eg modified bispecific antibodies). In some embodiments, modified antibodies (eg, bispecific or multispecific antibodies) bind to exemplary targets described elsewhere herein.

In some embodiments, the amino acid at position 91 (eg, the original amino acid) is substituted with Y to obtain a modified antibody (eg, a modified bispecific antibody). In some embodiments, the amino acid at position 94 (eg, the original amino acid) is substituted with Y to obtain a modified antibody (eg, a modified bispecific antibody). In some embodiments, the amino acid at position 96 (eg, the original amino acid) is substituted with W to obtain a modified antibody (eg, a modified bispecific antibody). In some embodiments, the amino acid at position 91 (eg, the original amino acid) is substituted with Y, and the amino acid at position 94 (eg, the original amino acid) is substituted with Y to obtain a modified antibody (eg, Modified bispecific antibody). In some embodiments, the amino acid at position 91 (eg, the original amino acid) is substituted with Y, and the amino acid at position 96 (eg, the original amino acid) is substituted with W to obtain a modified antibody (eg, Modified bispecific antibody). In some embodiments, the amino acid at position 94 (eg, the original amino acid) is substituted with Y, and the amino acid at position 96 (eg, the original amino acid) is substituted with W to obtain a modified antibody (eg, Modified bispecific antibody). In some embodiments, the amino acid at position 91 (eg, the original amino acid) is substituted with Y, the amino acid at position 94 (eg, the original amino acid) is substituted with Y, and the amino acid at position 96 (eg original amino acid) is substituted with W to obtain a modified antibody (eg modified bispecific antibody).

In some embodiments, the method further comprises substituting the amino acid at position 95 (e.g., the original amino acid) of the heavy chain variable domain ( _VH ) from an uncharged residue to a group selected from aspartic acid ( D), charged residues of arginine (R), glutamic acid (E) and lysine (K) to obtain modified antibodies (such as modified bispecific antibodies), wherein the amino acid numbers Department according to Kabat. In some embodiments, the method further comprises substituting the amino acid at position 95 (e.g., the original amino acid) of the heavy chain variable domain ( _VH ) from a non-aromatic residue to one selected from tryptophan ( W), aromatic residues of phenylalanine (F) and tyrosine (Y) to obtain modified antibodies (eg modified bispecific antibodies).

In some embodiments, methods of making (such as modifying or engineering) antibodies, such as bispecific antibodies, include modifying the _VH and/or _VL , for example, by substituting one or more of the above-discussed Or introduced into _VH and/or _VL to obtain modified _VH and/or modified _VL , and grafting modified _VH and/or modified _VL to antibodies (such as bispecific antibodies ) to obtain modified antibodies (such as modified bispecific antibodies).

In some embodiments, a _VH /VL _pair substituted, modified and/or engineered according to the methods described herein is subjected to at least one affinity maturation step (e.g., 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10 or more than 10 affinity maturation steps). Affinity maturation is a protocol for subjecting heavy chain/light chain pairs of antibodies, e.g. obtained by the methods described herein, to selection for increased affinity for a target (e.g. a target ligand or a target antigen, as described in further detail below) (see Wu et al. (1998) Proc Natl Acad Sci USA . 95, 6037-42). Details on affinity maturation of antibodies are also described in e.g. Merchant et al. (2013) Proc Natl Acad Sci US A. 110(32): E2987-96; Julian et al. (2017) Scientific Reports. 7: 45259; Tiller et al. (2017) Front. Immunol. 8: 986; Koenig et al. (2017) Proc Natl Acad Sci US A. 114(4): E486-E495; Yamashita et al. (2019) Structure. 27, 519-527; Payandeh et al. (2019) J Cell Biochem. 120: 940-950; Richter et al. (2019) mAbs. 11(1): 166-177; and Cisneros et al. (2019) Mol. Syst. Des. Eng. 4: 737-746 middle. In certain embodiments, one or more amino acid positions in the _VH and/or _VL of a heavy chain/light chain pair obtained by the methods herein are randomized (i.e. Those mentioned, ie at positions other than position 91, 94 and/or 96 in _{the VL} and optionally position 95 in the _VH ) to generate libraries of heavy chain/light chain variants. The library of _VH / _VL variants is then screened to identify those variants with the desired affinity for the target. Accordingly, in certain embodiments, the methods described herein further comprise the step of: (a) making the CDR-H1, CDR-H2, CDR-H3, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and/or CDR-L3 are mutagenized or randomized at one or more positions to generate a library of _VH / _VL variants, (b) making a library of _VH / _VL variants Contacting a target (eg, a target ligand or a target antigen), (c) detecting binding of the target to the _VH / _VL variant, and (d) obtaining a _VH /VL variant that specifically binds the target V _L variant. As mentioned above, positions 91, 94 and/or 96 in the _VL and optionally position 95 in the _VH in the antigen binding domain variants were not targeted for further randomization. Methods for mutagenizing the CDR-H1 , CDR-H2, CDR-H3, CDR-L1 , CDR-L2 and/or CDR-L3 of antibodies (or antigen-binding fragments thereof) are known in the art, and discussed elsewhere in this article. Details regarding libraries and library screening are provided elsewhere herein.

In certain embodiments, the methods described herein are further steps of: (e) determining the nucleic acid that specifically binds the _VH / _VL variant (i.e., the affinity matured _VH / _VL pair) of the target sequence. In some embodiments, the methods described herein further comprise the step of: (f) grafting an affinity matured _VH / _VL pair onto an antibody (such as a bispecific antibody) to obtain an affinity matured modified antibody (e.g. Affinity matured modified bispecific antibodies). In some embodiments, the methods described herein further comprise the step of: (g) assessing the extent to which affinity matured _VH / _{VL pairs} exhibit preferential pairing/preferential assembly, eg, using the methods described below.

Also provided herein are antibodies (eg, monospecific, bispecific, or multispecific antibodies) or antibody fragments produced according to any one or combination of the methods described above. Preferential pairing / preferential assembly of antibody heavy and light chains

As mentioned above, preferential pairing describes when pairing occurs between a first polypeptide and a second polypeptide while one or more additional different polypeptides (e.g., one or more additional heavy and/or light chains) are present Pairing pattern of a first polypeptide (such as a heavy chain) with a second polypeptide (such as a light chain). When HC ₁ is co-expressed with at least LC ₁ and LC ₂ , if the amount of HC ₁ /LC ₁ heavy chain-light chain pairing is greater than the amount of HC ₁ /LC ₂ pairing, in e.g. an antibody (e.g. a bispecific antibody) Preferential pairing (eg, homologous pairing) occurs between HC ₁ and LC ₁ . Likewise, when HC ₂ is co-expressed with at least LC ₁ and LC ₂ , if the amount of HC ₂ /LC ₂ heavy chain-light chain pairing is greater than the amount of HC ₂ /LC ₁ pairing, e.g. in a multispecific antibody (e.g. Preferential pairing (eg, homologous pairing) occurs between the HC ₂ and LC ₂ of a bispecific antibody). As described in further detail elsewhere herein, HC ₁ /LC ₁ , HC ₁ /LC ₂ , HC ₂ /LC ₁ , and HC ₂ /LC ₂ pairings can be determined by methods known in the art, such as liquid chromatography mass spectrometry ( LCMS) measurement.

In certain embodiments, the methods provided herein are used to generate (such as produce) antibodies (eg, bispecific antibodies) in which HC ₁ is preferentially paired with LC ₁ . Additionally or alternatively, the methods provided herein are used to generate (such as produce) antibodies (eg, bispecific antibodies) in which HC ₂ is preferentially paired with LC ₂ . In certain embodiments, _the methods provided herein are used to generate (such as produce) antibodies (eg, bispecific antibodies) in which HC ₁ is preferentially paired with LC 1 and HC ₂ is preferentially paired with LC ₂ . In certain embodiments, when HC ₁ is co-expressed with HC ₂ , LC ₁ , and LC ₂ of an antibody (eg, a bispecific antibody) produced by the methods provided herein, the desired pairing (eg, HC ₁ / Bispecific antibodies of LC ₁ and HC ₂ /LC ₂ ) are produced in relative yields of at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55% , at least about 60%, at least about 70%, at least about 71%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77% , at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87% , at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97% , at least about 99%, or greater than about 99%, including any range between such values. As described in the Examples, the relative yield of bispecific antibodies comprising the desired pairing (eg HC ₁ /LC ₁ and HC ₂ /LC ₂ ) can be determined using eg mass spectrometry.

In certain embodiments, expressed polypeptides of antibodies (such as bispecific antibodies) generated using the methods provided herein assemble with improved specificity to reduce the generation of mismatched heavy and light chains. In certain embodiments, the VH domain of _CH1 of an antibody provided herein (eg, a bispecific antibody) is assembled (such as preferentially assembled) with the _VL domain _{of LC1} during production. Method for assessing correct pairing / preferred pairing / preferred assembly

The preferential pairing, correct pairing, and/or preferential assembly of HC ₁ and LC ₁ of modified antibodies (e.g., modified bispecific antibodies) produced according to the methods described herein can be performed using methods well known to those of ordinary skill in the art. Determined in any of a number of ways. For example, the degree of preferential pairing of HC ₁ and LC ₁ in a modified antibody such as a modified bispecific antibody can be determined by light chain competition assay (LCCA). International Patent Application PCT/US2013/063306, filed October 3, 2013, describes various embodiments of LCCA and is hereby incorporated by reference in its entirety for all purposes. The method allows quantitative analysis of the pairing of heavy chains with specific light chains within a mixture of co-expressed proteins and can be used to determine whether a specific immunoglobulin heavy chain is associated with two immunoglobulin light chains when heavy and light chains are co-expressed One of them selectively associates. The method is briefly described as follows: at least one heavy chain and two different light chains are co-expressed in a cell in a ratio such that the heavy chain is a limited pairing reactant; the secreted protein is optionally isolated from the cell; binding to the heavy chain The immunoglobulin light chain polypeptide is separated from the remainder of the secreted protein to generate a separated heavy chain pair portion; detecting the amount of each distinct light chain in the separated heavy chain portion; and analyzing the separated heavy chain The relative amounts of each of the different light chains in the fraction were used to determine the ability of at least one heavy chain to pair selectively with one of the light chains.

In certain embodiments, the preferential pairing of HC ₁ and LC ₁ of a modified antibody (e.g., a modified bispecific or multispecific antibody) produced according to the methods provided herein is determined by mass spectrometry, such as liquid chromatography. chromatography-mass spectrometry (LC-MS), native mass spectrometry, acid mass spectrometry, etc.) measurements. Mass spectrometry was used to quantify the relative heterodimer populations comprising each light chain using the difference in their molecular weights to identify each distinct species. In certain embodiments, correct or preferential pairings are determined by LC-MS as described herein. In certain embodiments, the correct or preferential pairing of Fv or Fab is measured. Multispecific Antibody Format

Modified antibodies produced according to the methods provided herein, such as modified bispecific antibodies, can be used in any of a variety of bispecific or multispecific antibody formats known in the art. Numerous formats have been developed in the art to address the therapeutic opportunities offered by molecules with multiple binding specificities. Several methods have been described to prepare bispecific antibodies in which a specific antibody light chain or fragment is paired with a specific antibody heavy chain or fragment.

For example, mutations that promote selective pairing of cognate Fab or HC and LC pairing at the _CH1 / _CL interface are described in Dillon et al. (2017) MABS 9(2): 213-230 and WO2016/172485 , the content of which is incorporated herein by reference in its entirety.

Knob is a technique for heterodimerization of _CH3 domains of antibodies. Previously, the pestle technique has been applied to produce human full-length bispecific antibodies with a single common light chain (LC) (Merchant et al. "An efficient route to human bispecific IgG." Nat Biotechnol. 1998; 16:677-81; Jackman et al. "Development of a two-part strategy to identify a therapeutic human bispecific antibody that inhibits IgE receptor signaling." J Biol Chem. 2010;285:20850-9). See also WO1996027011, which is hereby incorporated by reference in its entirety for all purposes.

Antibodies generated using the methods provided herein, such as bispecific antibodies, can be further modified to include one or more additional heterodimerization domains that have a stronger preference for forming heterodimers over homodimers . Illustrative examples include, but are not limited to, eg WO2007147901 (Kjærgaard et al - Novo Nordisk: describes ionic interactions); WO 2009089004 (Kannan et al - Amgen: describes electrostatic steering); WO 2010/034605 (Christensen et al - Genentech; describes curly spiral). See also, for example, Pack, P. and Plückthun, A., Biochemistry 31, 1579-1584 (1992), describing the leucine zipper; or Pack et al., Bio/Technology 11, 1271-1277 (1993), describing the helix-turn - Spiral primitives. The phrases "heteromultimerization domain" and "heterodimerization domain" are used interchangeably herein. In certain embodiments, antibodies (such as bispecific antibodies) produced using the methods provided herein comprise one or more heterodimerization domains.

U.S. Patent Publication No. 2009/0182127 (Novo Nordisk, Inc.) describes the production of bispecific antibodies by modifying amino acid residues at the Fc interface and at the _CH1 : _CL interface of a light-heavy chain pair , which reduces the ability of the light chains of one pair to interact with the heavy chains of the other pair.

Techniques for making multispecific antibodies include, but are not limited to, the recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983)) and "knob and mortar". "Engineered (see, eg, US Pat. No. 5,731,168 and Atwell et al., J. Mol. Biol. 270:26-35 (1997)). Multispecific antibodies can also be produced by: engineering electrostatic steering for the production of antibody Fc-heterodimerized molecules (see e.g. WO 2009/089004); crossing two or more antibodies or fragments; (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); and the use of leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148( 5): 1547-1553 (1992) and WO 2011/034605).

Multispecific antibodies may also be provided in an asymmetric format with domain swapping in one or more binding arms of the same antigen specificity, i.e. by swapping _VH / _VL domains (see e.g. WO 2009/080252 and WO 2015/150447), _CH 1/C ₎ domains (see eg WO 2009/080253) or complete Fab arms (see eg WO 2009/080251, WO 2016/016299, see also Schaefer et al., PNAS, 108 ( 2011) 1187-1191 and Klein et al., MAbs 8 (2016) 1010-20). In one aspect, the multispecific antibody comprises crossed Fab fragments. The term "crossover Fab fragment" or "xFab fragment" or "exchange Fab fragment" refers to a Fab fragment in which the variable or constant regions of the heavy and light chains are exchanged. The crossover Fab fragment comprises a polypeptide chain consisting of a light chain variable region (V _L ) and a heavy chain constant region 1 ( _CH 1 ), and a polypeptide chain consisting of a heavy chain variable region (V _H ) and a light chain constant region ( _CL ). composed of polypeptide chains. Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations into domain interfaces to direct correct Fab pairing. See eg WO 2016/172485.

A review of various bispecific and multispecific antibody formats is provided in Klein et al., (2012) mAbs 4:6, 653-663 and Spiess et al. (2015) "Alternative molecular formats and therapeutic applications for bispecific antibodies." Mol. Immunol. 67 (2015) 95-106.

In some embodiments, modified antibodies (e.g., modified bispecific antibodies) produced by the methods provided herein are rearranged into any of the multispecific antibody formats described above to further Ensure correct heavy/light chain pairing. Production and purification of antibodies Culturing host cells

In certain embodiments, modified antibodies produced according to the methods provided herein, such as modified bispecific or multispecific antibodies, can be produced by (a) combining a set of antibodies encoding HC ₁ , The polynucleotides of _HC2 , _LC1 and _LC2 are introduced into host cells; and (b) culturing the host cells to produce antibodies (eg, bispecific or multispecific antibodies). In certain embodiments, polynucleotides encoding LC ₁ and LC ₂ are introduced into the host cell in a predetermined ratio (eg, molar ratio or weight ratio). In certain embodiments, the polynucleotides encoding LC ₁ and LC ₂ are introduced into the host cell such that the ratio (e.g., molar or weight ratio) of LC ₁ :LC ₂ is about 1:1, about 1:1: 1.5, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, about 1:5, about 1:5.5, about 1.5:1, about 2: 1. About 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1 or about 5.5:1, including any range between these values. In certain embodiments, the ratios are molar ratios. In certain embodiments, the ratios are by weight. In certain embodiments, polynucleotides encoding HC ₁ and HC ₂ are introduced into the host cell in a predetermined ratio (eg, molar ratio or weight ratio). In certain embodiments, the polynucleotides encoding HC ₁ and HC ₂ are introduced into the host cell such that the ratio (e.g., molar or weight ratio) of HC ₁ :HC ₂ is about 1:1, about 1:1 1.5, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, about 1:5, about 1:5.5, about 1.5:1, about 2: 1. About 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1 or about 5.5:1, including any range between these values. In certain embodiments, the ratios are molar ratios. In certain embodiments, the ratios are by weight. In certain embodiments, polynucleotides encoding HC ₁ , HC ₂ , LC ₁ , and LC ₂ are introduced into host cells in predetermined ratios (eg, molar or weight ratios). In certain embodiments, polynucleotides encoding HC ₁ , HC ₂ , LC ₁ and LC ₂ are introduced into host cells such that the ratio of HC ₁ + HC ₂ :LC ₁ + LC ₂ (e.g., molar ratio or weight ratio) is about 5:1, about 5:2, about 5:3, about 5:4, about 1:1, about 4:5, about 3:5, about 2:5 or about 1:5, including Any range between these values. In certain embodiments, polynucleotides encoding LC ₁ , LC ₂ , HC ₁ , and HC ₂ are introduced into host cells such that the ratio of LC ₁ +LC ₂ :HC ₁ +HC ₂ (e.g., molar ratio or Weight ratio) is about 1:1:1:1, about 2.8:1:1:1, about 1.4:1:1:1, about 1:1.4:1:1, about 1:2.8:1:1, about 1:1:2.8:1, about 1:1:1.4:1, about 1:1:1:2.8, or about 1:1:1:1.4, including any range between these values. In certain embodiments, the ratios are molar ratios. In certain embodiments, the ratios are by weight.

In certain embodiments, producing a modified antibody (such as a modified bispecific or multispecific antibody) produced according to the methods provided herein further comprises determining the optimal polynucleotide for introduction into the cell. ratio. In certain embodiments, mass spectrometry is used to determine antibody yields, such as bispecific antibody yields, and optimal chain ratios are adjusted to maximize protein yields, such as bispecific antibody yields. In certain embodiments, producing an antibody (such as a bispecific or multispecific antibody) produced according to the methods provided herein further comprises collecting or recovering the antibody from the cell culture. In certain embodiments, producing antibodies (such as bispecific or multispecific antibodies) produced according to the methods provided herein further comprises purifying the collected or recovered antibodies.

Host cells used to produce modified antibodies, such as modified bispecific antibodies, made according to the methods provided herein can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma)), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) is suitable for culturing host cells. Additionally, Ham et al., Meth. Enz . 58:44 (1979); Barnes et al., Anal. Biochem . 102:255 (1980); U.S. Pat. Any of the media described in No. 5,122,469; WO 90/03430; WO 87/00195; or US Patent Reference 30,985 can be used as a culture medium for host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™ drugs), trace elements (defined as inorganic compounds usually present in final concentrations in the micromolar range), and glucose or equivalent energy. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, and the like, are those previously used with host cells selected for expression and will be apparent to those of ordinary skill in the art. Collect or recover and purify antibodies

In a related aspect, producing a modified antibody (such as a modified bispecific antibody) produced according to the methods described herein comprises culturing the host cells described above under conditions that allow expression of the modified antibody and recovering (such as harvesting) the modified antibody. In certain embodiments, producing a modified antibody (such as a modified bispecific antibody) produced according to the methods described herein further comprises purifying the recovered modified antibody (such as a modified bispecific antibody) A substantially homogeneous preparation is obtained, eg for further testing and use.

Modified antibodies produced according to the methods described herein, such as modified bispecific antibodies, can be produced intracellularly, or secreted directly into the culture medium. If such modified antibodies are produced intracellularly, specific debris, ie host cells or lysed fragments, are removed as a first step, for example by centrifugation or ultrafiltration. Where modified antibodies produced according to the methods described herein, such as modified bispecific antibodies, are secreted into the culture medium, they are generally first concentrated using commercially available protein concentration filters, such as Amicon or Millipore Pellicon ultrafiltration units. Supernatants from such expression systems. A protease inhibitor such as PMSF may be included in any of the preceding steps to inhibit proteolysis and an antibiotic may be included to prevent growth of exogenous contaminants.

Standard protein purification methods known in the art can be employed to obtain substantially homogeneous preparations of modified antibodies, such as modified bispecific antibodies, produced from cells according to the methods described herein. The following procedures illustrate suitable purification procedures: fractionation on immunoaffinity or ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or on cation exchange resins such as DEAE, chromatofocusing, SDS-PAGE , ammonium sulfate precipitation and gel filtration using eg Sephadex G-75.

Additionally or alternatively, modified antibodies (such as modified bispecific antibodies) produced using the methods described herein can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, wherein Affinity chromatography is the preferred purification technique.

In certain aspects, a formulation derived from a cell culture medium as described above is applied to a protein A immobilized solid phase to allow specific binding of a modified antibody, such as a modified bispecific antibody, to protein A . The solid phase is then washed to remove contaminants that are non-specifically bound to the solid phase. Modified antibodies, such as modified bispecific antibodies, are recovered from the solid phase by elution into a solution containing a chaotropic agent or a mild detergent. Exemplary chaotropic agents and mild detergents include, but are not limited to, guanidine hydrochloride, urea, lithium perchlorate, arginine, histidine, SDS (sodium dodecyl sulfate), Tween, Triton, and NP-40 , all of which are commercially available.

The suitability of protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody, such as a bispecific antibody. Protein A can be used to purify antibodies based on human γ1, γ2 or γ4 heavy chains (Lindmark et al., J. Immunol. Meth . 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human γ3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanistically stable matrices such as controlled pore glass or poly(styrene divinyl)benzene allow faster flow rates and shorter processing times than can be achieved with agarose. In cases where the modified antibody (such as a modified bispecific antibody) comprises a _CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is suitable for purification. Depending on the antibody to be recovered (such as a bispecific antibody), other techniques for protein purification such as fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica, on heparin SEPHAROSE Chromatography on ™, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also useful.

After one or more of any preliminary purification steps, the mixture comprising the modified antibody (such as a modified bispecific antibody) and contaminants can be subjected to elution using an elution buffer with a pH between about 2.5-4.5. Low pH HIC is preferably performed at low salt concentrations (eg, about 0-0.25M salt). Production of modified antibodies, such as modified bispecific antibodies, alternatively or additionally (for any of the aforementioned specific methods) comprises dialysis of a solution comprising a mixture of polypeptides. Libraries and Library Screening

Also provided herein are libraries of heavy chain/light chain pairs (or antigen-binding fragments thereof) exhibiting preferential pairing.

For example, provided herein is a library comprising a plurality of antigen binding domain variants, each antigen binding domain variant comprising a different antibody heavy chain domain ( _VH ) and a different antibody light chain domain ( _VL ), wherein Each _VH comprises a different CDR-H1, CDR-H2 and CDR-H3 sequence, wherein each _VL comprises a different CDR-L1, CDR-L2 and CDR-L3 sequence, and wherein position 94 in each _VL or each _VL At least one amino acid at position 96 is a charged residue selected from aspartic acid (D), arginine (R), glutamic acid (E) and lysine (K), wherein the amino acid Numbering is according to Kabat. In some embodiments, both amino acids at positions 94 and 96 of each _VL are charged residues independently selected from D, R, E, and K. In some embodiments, the amino acid at position 94 of each _VL is D. In some embodiments, the amino acid at position 96 of each _VL is R. In some embodiments, the amino acid at position 94 of each _VL is D and the amino acid at position 96 of each _VL is R. In some embodiments, the amino acid at position 95 of each _VH is a charged residue selected from D, R, E, and K. In some embodiments, the amino acid at position 95 of each _VH is D. In some embodiments, the amino acid at position 94 of each _VL is D, the amino acid at position 96 of each _VL is R, and the amino acid at position 95 of each _VH is D.

Also provided herein is a library comprising a plurality of antigen binding domain variants, each antigen binding domain variant comprising a different antibody heavy chain domain ( _VH ) and a different antibody light chain domain ( _VL ), wherein each _VH Comprising different CDR-H1, CDR-H2 and CDR-H3 sequences, wherein each V _L comprises different CDR-L1, CDR-L2 and CDR-L3 sequences, and wherein position 91 of each V _L , position 94 in each V _L Or at least one amino acid at position 96 of each _VL is an aromatic residue selected from tryptophan (W), phenylalanine (F) and tyrosine (Y), wherein the amino acid numbering is according to Kabat . In some embodiments, at least two amino acids at position 91, position 94, or position 96 (e.g., positions 91 and 94, positions 91 and 96, or positions 94 and 96) of each _VL are selected from W, F, and Aromatic residue of Y. In some embodiments, the amino acid at position 91 of each _VL is Y. In some embodiments, the amino acid at position 94 of each _VL is Y. In some embodiments, the amino acid at position 96 of each _VL is W. In some embodiments, the amino acid at position 91 of each _VL is Y, and the amino acid at position 94 of each _VL is Y. In some embodiments, the amino acid at position 91 of each _VL is Y, and the amino acid at position 96 of each _VL is W. In some embodiments, the amino acid at position 94 of each _VL is Y, and the amino acid at position 96 of each _VL is W. In some embodiments, the amino acid at position 91 of each _VL is Y, the amino acid at position 94 of each _VL is Y, and the amino acid at position 96 of each _VL is W. In some embodiments, the amino acid at position 95 of each _VH is a charged amino acid selected from aspartic acid (D), arginine (R), glutamic acid (E) and lysine (K). residues, where amino acid numbering is according to Kabat. In some embodiments, the amino acid at position 95 of each _VH is an aromatic residue selected from tryptophan (W), phenylalanine (F), and tyrosine (Y).

In certain embodiments, the library is a library of polypeptides (such as any of a plurality of polypeptides described herein). In certain embodiments, the polypeptide libraries provided herein are polypeptide display libraries. Such polypeptide display libraries can be screened to select and/or elicit binding proteins with desired properties for a wide variety of utilities including, but not limited to, therapeutic, prophylactic, veterinary, diagnostic, reagent or material applications. In certain embodiments, the library is a library of nucleic acids (such as any of a plurality of nucleic acids described herein), wherein each nucleic acid (or set of nucleic acids) encodes a different antigen domain binding variant described herein. In some embodiments, a library is a plurality of host cells (e.g., prokaryotic or eukaryotic host cells) each comprising (and, for example, expressing) a different nucleic acid (or set of nucleic acids), wherein each different nucleic acid (or set of nucleic acids) encodes a nucleic acid herein Said different antigenic domain binding variants.

In certain embodiments, the libraries provided herein comprise at least 2, 3, 4, 5, 10, 30, 100, 250, 500, 750, 1000, 2500, 5000, 7500, 10000, 25000, 50000, 75000, 100000, 250000, 500000, 750000, 1000000, 2500000, 5000000, 7500000, 10000000 or more than 10000000 different antigen binding domains, including any range between such values. In certain embodiments, the libraries provided herein have about 2, about 5, about 10, about 50, about 100, about 250, about 500, about 750, about 10 ³ , about 10 ⁴ , about 10 ⁵ , about 10 ⁶ , about 10 ⁷ , about 10 ⁸ , about 10 ⁹ , about 10 ¹⁰ , about 10 ¹¹ , about 10 ¹² , about 10 ¹³ , about 10 ¹⁴ or more than about 10 ¹⁴ (such as about 10 ¹⁵ or about 10 ¹⁶ ) The sequence diversity of , including any range between these values.

In certain embodiments, the libraries provided herein are generated through genetic engineering. Various methods for mutagenesis and subsequent library construction (and appropriate methods for screening or selection) have been described previously. Such mutagenesis methods include, but are not limited to, eg error-prone PCR, loop shuffling or oligonucleotide site-directed mutagenesis, random nucleotide insertion or other methods prior to recombination. Further details on these methods are described in, for example, Abou-Nadler et al. (2010) Bioengineered Bug s 1, 337-340; Firth et al. (2005) Bioinformatics 21, 3314-3315; Cirino et al. (2003) Methods Mol Biol 231 , 3-9; Pirakitikulr (2010) Protein Sci 19, 2336-2346; Steffens et al. (2007) J. Biomol Tech 18, 147-149; and elsewhere. Accordingly, in certain embodiments, multispecific antigen-binding protein libraries generated through genetic engineering techniques are provided.

In certain embodiments, the libraries provided herein are generated via in vitro translation. Briefly, in vitro translation entails colonizing one or more protein-coding sequences into a vector containing a promoter, producing mRNA by transcribing the one or more colonized sequences with RNA polymerase, and by, for example, using Cell-free extracts translate this mRNA into protein in vitro. Desired mutant proteins can be produced simply by altering the cloned protein coding sequence. Many mRNAs are efficiently translated in wheat germ extract or in rabbit reticulocyte lysates. Further details on in vitro translation are described, for example, in Hope et al. (1985) Cell 43, 177-188; Hope et al. (1986) Cell 46, 885-894; Hope et al. (1987) EMBO J. 6, 2781-2784 ; Hope et al. (1988) Nature 333, 635-640; and Melton et al. (1984) Nucl. Acids Res. 12, 7057-7070.

Accordingly, there is provided a plurality of nucleic acid molecules encoding the polypeptide display libraries described herein. Also provided herein are expression vectors operably linked to a plurality of nucleic acid molecules. Also provided is a method of making a library provided herein by providing a plurality of nucleic acids encoding a plurality of antigen binding domains described herein and expressing the nucleic acids.

In certain embodiments, the libraries provided herein are generated via chemical synthesis. Methods of solid-phase and solution-phase peptide synthesis are well known in the art and are described in detail in, for example, Methods of Molecular Biology, 35 , Peptide Synthesis Protocols, (eds. MW Pennington and BM Dunn), Springer, 1994; Welsch et al. ( 2010) Curr Opin Chem Biol 14, 1-15; Methods of Enzymology, 289 , Solid Phase Peptide Synthesis, (Edited by GB Fields), Academic Press, 1997; Chemical Approaches to the Synthesis of Peptides and Proteins, (P. Lloyd-Williams , F. Albericio and E. Giralt), CRC Press, 1997; Fmoc Solid Phase Peptide Synthesis, A Practical Approach, (WC Chan, PD White), Oxford University Press, 2000; Solid Phase Synthesis, A Practical Guide, ( SF Kates, F Albericio, eds), Marcel Dekker, 2000; P. Seneci, Solid-Phase Synthesis and Combinatorial Technologies, John Wiley & Sons, 2000; Synthesis of Peptides and Peptidomimetics (M. Goodman, Editor-in-chief, A. Felix, L. Moroder, C. Tmiolo eds), Thieme, 2002; NL Benoiton, Chemistry of Peptide Synthesis, CRC Press, 2005; Methods in Molecular Biology, 298, Peptide Synthesis and Applications, (J. Howl eds) Humana Press, 2005; and Amino Acids, Peptides and Proteins in Organic Chemistry, Volume 3, Building Blocks, Catalysts and Coupling Chemistry, (ed. AB Hughs) Wiley-VCH, 2011. Accordingly, in certain embodiments, libraries of multispecific antigen-binding proteins produced via chemical synthesis techniques are provided.

In certain embodiments, the libraries provided herein are presentation libraries. In certain embodiments, the display library is a phage display library, a phagemid display library, a virus display library, a bacterial display library, a yeast display library, a lambda gt11 library, a CIS display library, and an in vitro compartmentalized library or a ribosome display library . Methods for making and screening such presentation libraries are well known to those skilled in the art and are described, for example, in Molek et al. (2011) Molecules 16, 857-887; Boder et al., (1997) Nat Biotechnol 15, 553-557; Scott et al. (1990) Science 249, 386-390; Brisette et al. (2007) Methods Mol Biol 383, 203-213; Kenrick et al. (2010) Protein Eng Des Sel 23, 9-17; Freudl et al. (1986) J Mol Biol 188, 491-494; Getz et al (2012) Methods Enzymol 503, 75-97; Smith et al (2014) Curr Drug Discov Technol 11, 48-55; Hanes et al (1997) Proc Natl Acad Sci USA 94 , 4937-4942; Lipovsek et al., (2004) J Imm Methods 290, 51-67; Ullman et al. (2011) Brief. Funct. Genomics, 10, 125-134; Odegrip et al. (2004) Proc Natl Acad Sci USA 101, 2806-2810; and in Miller et al. (2006) Nat Methods 3, 561-570.

In certain embodiments, the libraries provided herein are RNA protein fusion libraries generated, eg, by the techniques described in US 6258558, US 6261804, US 5643768 and US 5658754 by Szostak et al. In certain embodiments, the libraries provided herein are DNA protein libraries such as described in US6416950. screening method

Libraries provided herein can be screened to identify antigen-binding variants with high affinity for a target (eg, antigen) of interest. Accordingly, provided herein is a method of obtaining an antigen-binding variant that binds a target of interest, such as a target of interest described elsewhere herein.

In certain embodiments, the method comprises a) contacting a library described herein with an antigen binding domain variant in the library that specifically binds the target under conditions that permit binding of the target of interest, (b) detecting binding of the target to the antigen-binding domain variant that specifically binds the target (e.g., detecting a complex comprising the target and the antigen-binding domain variant that specifically binds the target), and (c) Antigen-binding domain variants that specifically bind the target are obtained. In _some embodiments, the method further comprises subjecting the antigen binding domain variant thus identified to at least one affinity maturation step, wherein position 91, position 94 and/or The amino acid at position 96 was used for randomization. In some embodiments, the amino acid at position 95 in the _VH is not selected for randomization.

In some embodiments, the method further comprises producing an antibody (such as a bispecific antibody) comprising an antigen binding domain variant that binds the target of interest (e.g., an affinity matured antigen binding domain variant that binds the target of interest). antibodies or multispecific antibodies).

In certain embodiments, complexes comprising a target and an antigen binding domain variant that specifically binds to the target are provided. In certain embodiments, the method further comprises determining one or more nucleic acid sequences of the _VH and/or _VL of the antigen-binding domain variant.

Affinity maturation is the process of subjecting an antigen-binding domain variant to a protocol selected for increased affinity to a target (eg, target ligand or target antigen) (see Wu et al. (1998) Proc Natl Acad Sci USA . 95, 6037-42). In certain embodiments, antigen binding domain variants that specifically bind the first target ligand are further randomized after identification from library screening (i.e., in addition to those mentioned above, That is, at positions other than positions 91, 94 and/or 96 in _VL and optionally position 95 in _VH ). For example, in certain embodiments, the method of obtaining an antigen binding domain variant that specifically binds a first target ligand further comprises (e) making the CDRs of a previously identified antigen binding domain variant -H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and/or CDR-L3 mutagenesis or randomization to generate further antigen binding domain variants, (f) make the first target ligand Contacting the further randomized antigen binding domain variant, (g) detecting binding of the target to the further randomized antigen binding domain variant, and (h) obtaining further randomized variants that specifically bind the target Antigen-binding domain variants. As mentioned above, positions 91, 94 and/or 96 in the _VL and optionally position 95 in the _VH in the antigen binding domain variants were not targeted for further randomization. Methods of mutagenizing the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and/or CDR-L3 of an antigen binding domain are known in the art and may include, for example, random Mutagenesis, CDR walking mutagenesis or sequential and parallel optimization, mutagenesis by structure-based rational design, site-specific mutagenesis, enzyme-based mutagenesis, chemical-based mutagenesis and for the synthesis of antibodies Gene synthesis method for gene production. See, eg, Yang et al., 1995, CDR Walking Mutagenesis for the Affinity Mutation of a Potent Human Anti-HIV-1 Antibody into the Picomolar Range, J. Mol. Biol. 254:392-40, and Lim et al., 2019, Review : Cognizance of Molecular Methods for the Generation of Mutagenic Phage Display Antibody Libraries for Affinity Maturation, Int. J. Mol. Sci, 20:1861, the contents of which are incorporated herein by reference in their entirety.

In certain embodiments, the method further comprises (i) determining the nucleic acid sequence of the antigen binding domain variant that specifically binds the target.

In certain embodiments, further randomized antigen binding domain variants comprise at least one or at least two randomized CDRs that were not previously randomized in the first library. Multiple rounds of randomization (i.e., with the exception of positions 91, 94, and/or 96 in the _VL and, optionally, position 95 in the _VH ), screening and selection can be performed until one or more with sufficient affinity for the target is obtained. variants of the antigen-binding domain. Thus, in certain embodiments, steps (e)-(h) or steps (e)-(i) are repeated once, twice, three times, four times, five times, six times, seven times, eight times, nine times times, ten times, or more than ten times to identify antigen binding domain variants that specifically bind one or more of the first target ligands. In some embodiments, one or more of the antigen-binding domain variants that have undergone two or more rounds of randomization, screening, and selection bind a target with an affinity at least as high as that that has undergone one of the rounds of randomization, screening, and selection or These affinities are generally high for multiple antigen binding domain variants.

Libraries of antigen binding domain variants described herein can be screened by any technique known in the art for eliciting new or improved binding proteins that specifically bind a target ligand. In certain embodiments, the target ligand is immobilized on a solid support, such as a column resin or well of a microtiter plate, and the target ligand is combined with a library of candidate multispecific antigen-binding proteins, such as any library described herein) contact. Selection techniques can be, for example, phage display (Smith (1985) Science 228, 1315-1317), mRNA display (Wilson et al. (2001) Proc Natl Acad Sci USA 98: 3750-3755), bacterial display (Georgiou et al. (1997) Nat Biotechnol 15:29-34.), yeast presentation (Boder and Wittrup (1997) Nat. Biotechnol . 15:553-5577) or ribosome presentation (Hanes and Plückthun (1997) Proc Natl Acad Sci USA 94:4937-4942 and WO2008/068637).

In certain embodiments, the library of antigen binding domain variants is a phage display library. In certain embodiments, phage particles displaying the antigen binding domain variants described herein are provided. In certain embodiments, phage particles displaying an antigen binding domain variant described herein capable of binding to a target ligand are provided.

Phage display is a technique in which multiple variants of a multispecific antigen-binding protein are presented as fusion proteins with a coat protein on the surface of a phage particle (Smith, GP (1985) Science , 228:1315-7; Scott, JK and Smith, GP (1990) Science 249: 386; Sergeeva, A. et al. (2006) Adv. Drug Deliv. Rev. 58:1622-54). The utility of phage display is that large libraries of selectively randomized protein variants (or randomly cloned cDNAs) can be sorted rapidly and efficiently against those sequences that bind to target molecules with high affinity.

Peptides (Cwirla, SE et al. (1990) Proc. Natl. Acad. Sci. USA , 87:6378) or proteins (Lowman, HB et al. (1991) Biochemistry , 30:10832; Clackson, T. et al. (1991) Nature , 352: 624; Marks, JD et al. (1991), J. Mol. Biol. , 222:581; Kang, AS et al. (1991) Proc. Natl. Acad. Sci. USA , 88:8363) library in Display on phage has been used to screen millions of polypeptides or oligopeptides for specific binding properties (Smith, GP (1991) Current Opin. Biotechnol ., 2:668; Wu et al. (1998) Proc Natl Acad Sci USA . May 95, 6037-42). Multivalent phage display methods have been used to display small random peptides and small proteins via fusions to gene III or gene VIII of filamentous phage. (Wells and Lowman, Curr. Opin. Struct. Biol. , 3:355-362 (1992) and references cited therein.) In monovalent phage display, the protein or peptide library is fused to gene III or a portion thereof, and Expression at low levels in the presence of wild-type gene III protein allows phage particles to display one copy of the fusion protein or none of the fusion protein. Avidity effects are reduced relative to multivalent phage to allow selection based on intrinsic ligand affinity and use of phagemid vectors that simplify DNA manipulation. (Lowman and Wells, Methods: A companion to Methods in Enzymology , 3:205-0216 (1991).)

Sorting phage libraries of antigen-binding domain variants requires the construction and propagation of large numbers of variants, procedures for affinity purification using target ligands, and a means of evaluating the results of binding enrichment (see e.g. US 5223409, US 5403484, US 5571689 and US 5663143).

Most phage display methods use filamentous phage (such as M13 phage). λ-shaped phage display system (see WO1995/34683, US 5627024), T4 phage display system (Ren et al. (1998) Gene 215:439; Zhu et al. (1998) Cancer Research , 58:3209-3214; Jiang et al., (1997) Infection & Immunity , 65:4770-4777; Ren et al. (1997) Gene , 195:303-311; Ren (1996) Protein Sci. , 5:1833; Efimov et al. (1995) Virus Genes , 10: 173) and the T7 phage display system (Smith and Scott (1993) Methods in Enzymology , 217: 228-257; US. 5766905) are also known.

Many other refinements and variations of the basic phage display concept have been developed. These improvements enhance the ability of the presentation system to screen peptide libraries for binding to selected target molecules and to present functional proteins to potentially screen them for desired properties. A combinatorial reaction setup for phage display reactions has been developed (WO 1998/14277) and phage display libraries have been used to analyze and control bimolecular interactions (WO 1998/20169; WO 1998/20159) and properties of constrained helical peptides ( WO 1998/20036). WO 1997/35196 describes a method for isolating affinity ligands, wherein a phage display library is contacted with one solution in which the ligand will bind to the target molecule and a second solution in which the affinity ligand will not bind to the target molecule, to Selective isolation of bound ligands. WO 1997/46251 describes a method of biopanning random phage display libraries with affinity purified antibodies, followed by isolation of binding phage, followed by a micropanning process using microtiter wells to isolate high affinity binding phage. This approach can be applied to the libraries of antigen binding domain variants disclosed herein. The use of Staphylococcus aureus protein A as an affinity tag has also been reported (Li et al. (1998) Mol Biotech. 9:187). WO 1997/47314 describes the use of substrate subtraction libraries to differentiate enzyme specificities using combinatorial libraries which may be phage display libraries. Additional methods of selecting specific binding proteins are described in US 5498538, US 5432018 and WO 1998/15833. Methods of generating peptide libraries and screening such libraries are also disclosed in US 5723286, US 5432018, US 5580717, US 5427908, US 5498530, US 5770434, US 5734018, US 5698426, US 5763192 and US 5723323. Exemplary Antigens / Target Molecules

Examples of molecules that can be targeted by antibodies (eg, bispecific or multispecific antibodies) produced using the methods provided herein include, but are not limited to, soluble serum proteins and their receptors and other membrane-bound proteins (eg, adhesins). In another embodiment, the multispecific antigen binding proteins provided herein are capable of binding one, two or more cytokines, cytokine-related proteins, and cytokine receptors selected from the group consisting of 8MPI, 8MP2, 8MP38 (GDFIO), 8MP4, 8MP6, 8MP8, CSFI (M-CSF), CSF2 (GM-CSF), CSF3 (G-CSF), EPO, FGF1 (αFGF), FGF2 (βFGF), FGF3 (int- 2), FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF9, FGF10, FGF11, FGF12, FGF12B, FGF14, FGF16, FGF17, FGF19, FGF20, FGF21, FGF23, IGF1, IGF2 , IFNA1, g1.

IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFN81, IFNG, IFNWI, FEL1, FEL1 (EPSELON), FEL1 (ZETA), IL 1A, IL 1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL1 0, IL 11, IL 12A, IL 12B, IL 13, IL 14, IL 15, IL 16, IL 17, IL 17B, IL 18, IL 19, IL20, IL22, IL23, IL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL30, PDGFA, PDGFB, TGFA, TGFB1, TGFB2, TGFBb3, LTA (TNF-β), LTB, TNF (TNF-α), TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand ), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1 BB ligand), TNFSF10 (TRAIL), TNFSF11 (TRANCE), TNFSF12 (APO3L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFSF15 (VEGI), TNFSF18, HGF (VEGFD), VEGF, VEFGA, VEGFB, VEGFC, IL1R1, IL1R2, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R , IL5RA, IL6R, IL7R, IL8RA, IL8RB, IL9R, IL10RA, IL10RB, IL 11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17R, IL18R1, IL20RA, IL21R, IL22R, IL1HY1, IL1RAP, IL1RAPL1, IL1RANPL, 2, IL1R IL6ST, IL18BP, IL18RAP, IL22RA2, AIF1, HGF, LEP (leptin), PTN and THPO.

In another embodiment, the target molecule is a chemokine, a chemokine receptor, or a chemokine-associated protein selected from the group consisting of: CCLI (1-309), CCL2 (MCP-1/MCAF) , CCL3 (MIP-Iα), CCL4 (MIP-Iβ), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CCL11 (eotaxin), CCL 13 (MCP- 4), CCL 15 (MIP-Iδ), CCL 16 (HCC-4), CCL 17 (TARC), CCL 18 (PARC), CCL 19 (MDP-3b), CCL20 (MIP-3α), CCL21 (SLC/ Secondary non-lymphoid chemoattractant-2 (exodus-2)), CCL22 (MDC / STC-1), CCL23 (MPIF-1), CCL24 (MPIF-2 / eosin-2), CCL25 (TECK ), CCL26 (Eosin-3), CCL27 (CTACK / ILC), CCL28, CXCLI (GROI), CXCL2 (GR02), CXCL3 (GR03), CXCL5 (ENA-78), CXCL6 (GCP-2), CXCL9 (MIG), CXCL 10 (IP 10), CXCL 11 (1-TAC), CXCL 12 (SDFI), CXCL 13, CXCL 14, CXCL 16, PF4 (CXCL4), PPBP (CXCL7), CX3CL 1 (SCYDI) , SCYEI, XCLI (lymphotactin), XCL2 (SCM-Iβ), BLRI (MDR15), CCBP2 (D6 / JAB61), CCRI (CKRI / HM145), CCR2 (mcp-IRB IRA), CCR3 (CKR3 / CMKBR3), CCR4, CCR5 (CMKBR5 / ChemR13), CCR6 (CMKBR6 / CKR-L3 / STRL22 / DRY6), CCR7 (CKR7 / EBII), CCR8 (CMKBR8 / TER1 / CKR-L1), CCR9 (GPR- 9-6), CCRL1 (VSHK1), CCRL2 (L-CCR), XCR1 (GPR5 / CCXCR1), CMKLR1, CMKOR1 (RDC1), CX3CR1 (V28), CXCR4, GPR2 (CCR10), GPR31, GPR81 (FKSG80), CXCR3 (GPR9/CKR-L2), CXCR6 (TYMSTR/STRL33 / Bonzo), HM74, IL8RA (IL8Rα), IL8RB (IL8Rβ), LTB4R (GPR16), TCP10, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSF8 , BDNF, C5R1, CSF3, GRCC10 (C10), EPO, FY (DARC), GDF5, HDF1, HDF1α, DL8, PRL, RGS3, RGS13, SDF2, SLIT2, TLR2, TLR4, TREM1, TREM2 and VHL.

In another embodiment, antibodies (e.g., bispecific or multispecific antibodies) produced using the methods provided herein are capable of binding one or more targets selected from the group consisting of: ABCF1; ACVR1; ACVR1B; ACVR2 ; ACVR2B; ACVRL1; ADORA2A; proteoglycan (Aggrecan); AGR2; AICDA; AIF1; AIG1; AKAP1; AKAP2; AMH; AMHR2; ANGPTL; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; a-glycoprotein); B7.1; B7.2; BAD; BAFF (BLys); BAG1; BAI1; BCL2; BCL6; BDNF; BLNK; BLRI (MDR15); BMP1; BMP2; BMP3B (GDF10); BMP4; BMP6 BMP8; BMPR1A; BMPR1B; BMPR2; BPAG1 (plectin); BRCA1; C19orf10 (IL27w); C3; C4A; C5; C5R1; CANT1; CASP1; CASP4; CAV1; CCBP2 (D6/JAB61); 1-309); CCL11 (eostatin); CCL13 (MCP-4); CCL15 (MIP1δ); CCL16 (HCC-4); CCL17 (TARC); CCL18 (PARC); CCL19 (MIP-3β); CCL2 (MCP-1); MCAF; CCL20 (MIP-3α); CCL21 (MTP-2); SLC; Secondary nonlymphoid chemoattractant-2; CCL22 (MDC/STC-1); CCL23 (MPIF-1) CCL24 (MPIF-2/Eosin-2); CCL25 (TECK); CCL26 (Eosin-3); CCL27 (CTACK / ILC); CCL28; CCL3 (MTP-Iα); CCL4 (MDP- CCL5 (RANTES); CCL7 (MCP-3); CCL8 (mcp-2); CCNA1; CCNA2; CCND1; CCNE1; CCNE2; CCR1 (CKRI / HM145); CCR2 (mcp-IRβ / RA); CCR3 ( CKR/CMKBR3); CCR4; CCR5 (CMKBR5 / ChemR13); CCR6 (CMKBR6/CKR-L3 / STRL22 / DRY6); CCR7 (CKBR7 / EBI1); CCR8 (CMKBR8 / TER1 / CKR-L1); -6); CCRL1 (VSHK1); CCRL2 (L-CCR); CD164; CD19; CD1C; CD20; CD200; CD22; CD24; CD28; CD3; CD37; CD38; CD3E; CD3G; CD3Z; CD4; CD40; CD40L; CD44; CD45RB; CD52; CD69; CD72; CD74; CD79A; CD79B; CDS; CD80; CD81; CD83; CD86; CDH1 (E-cadherin); CDH10; CDH12; CDH13; CDH18; CDH20; CDH5; CDH7; CDH8; CDH9; CDK2; CDK3; CDK4; CDK5; CDK6; CDK7; CDK9; CDKN1A (p21/WAF1/Cip1); CDKN1B (p27/Kip1); CDKN1C; ; CDKN3; CEBPB; CER1; CHGA; CHGB; Chitinase; CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6; CKLFSF7; CKLFSF8; CLDN3; CLN3; CLU (clusterin); CMKLR1; CMKOR1 (RDC1); CNR1; COL 18A1; COL1A1; COL4A3; COL6A1; CR2; CRP; CSFI (M-CSF); CSF2 (GM-CSF); ); CTLA4; CTNNB1 (b-catenin); CTSB (cathepsin B); CX3CL1 (SCYDI); CX3CR1 (V28); CXCL1 (GRO1); CXCL10 (IP-10); CXCL11 (I-TAC/IP-9); CXCL12 (SDF1); CXCL13; CXCL14; CXCL16; CXCL2 (GRO2); CXCL3 (GRO3); CXCL5 (ENA-78/LIX); CXCL6 (GCP-2); CXCR3 (GPR9/CKR-L2); CXCR4; CXCR6 (TYMSTR/STRL33/Bonzo); CYB5; CYC1; CYSLTR1; DAB2IP; DES; DKFZp451J0118; ; EGF; EGFR; ELAC2; ENG; ENO1; ENO2; ENO3; EPHB4; EPO; ERBB2 (Her-2); EREG; ERK8; ESR1; ESR2; F3 (TF); FGF; FGF1 (αFGF); FGF10; FGF11; FGF12; FGF12B; FGF13; FGF14; FGF16; FGF17; FGF18; FGF19; FGF2 (bFGF); FGF20; FGF5; FGF6 (HST-2); FGF7 (KGF); FGF8; FGF9; FGFR3; FIGF (VEGFD); FEL1 (EPSILON); FIL1 (ZETA); FLJ12584; FLJ25530; ); FLT1; FOS; FOSL1 (FRA-1); FY (DARC); GABRP (GABAa); GAGEB1; GAGEC1; GALNAC4S-6ST; GATA3; GDF5; GFI1; GGT1; GM-CSF; ); GPR31; GPR44; GPR81 (FKSG80); GRCCIO (C10); GRP; GSN (Gelsolin); GSTP1; HAVCR2; HDAC4; HDAC5; HDAC7A; HDAC9; HGF; HIF1A; HOP1; histamine and tissue Amine receptors; HLA-A; HLA-DRA; HM74; HMOXI; HUMCYT2A; ICEBERG; ICOSL; 1D2; IFN-a; IFNA1; IFNA2; IFNA4; IFNA5; IFNA6; IFNA7; IFNB1; IGF1R; IGF2; IGFBP2; IGFBP3; IGFBP6; IL-1; IL10; IL10RA; IL10RB; IL11; IL17; IL17B; IL17C; IL17R; IL18; IL18BP; IL18R1; IL18RAP; IL19; IL1A; IL1B; ILIF10; IL1F5; IL1F6; IL1F7; IL1F8; IL1F9; IL2; IL20; IL20RA; IL21 R; IL22; IL22R; IL22RA2; IL23; IL24; IL25; IL26; IL27; IL28A; IL28B; IL29; IL2RA; IL2RB; IL2RG; IL3; IL5RA; IL6; IL6R; IL6ST (glycoprotein 130); EL7; EL7R; EL8; IL8RA; DL8RB; IL8RB; DL9; DL9R; DLK; INHA; INHBA; INSL3; INSL4; ITGA6 (a6 integrin); ITGAV; ITGB3; ITGB4 (b4 integrin); JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR; KITLG; KLF5 (GC Box BP); KLF6 KLKIO; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (Keratin 19); KRT2A; KHTHB6 (hair-specific type H keratin)); LAMAS; LEP (leptin); Lingo-p75; Lingo-Troy; LPS; LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR; MACMARCKS; MAG or OMgp; MAP2K7 (c -Jun); MDK; MIB1; midkine; MEF; MIP-2; MKI67; (Ki-67); MMP2; MMP9; MS4A1; MSMB; MTSS1; MUC1 (mucin); MYC; MY088; NCK2; neurocan; NFKB1; NFKB2; NGFB (NGF); NGFR; NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75 ; NgR-Troy; NME1 (NM23A); NOX5; NPPB; NR0B1; NR0B2; NR1D1; NR1D2; NR1H2; NR1H3; NR1H4; NR4A1; NR4A2; NR4A3; NR5A1; NR5A2; NR6A1; NRP1; NRP2; NT5E; NTN4; ODZI; OPRD1; ; PGR; phosphacan; PIAS2; PIK3CG; PLAU (uPA); PLG; PLXDC1; PPBP (CXCL7); PPID; PRI; PRKCQ; PRKDI; PRL; PROC; PROK2; PSAP; PSCA; PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p21 Rac2); RARB; RGSI; RGS13; RGS3; RNF110 (ZNF144); ROBO2; S100A2; SCGB1D2 (lipophilin B); SCGB2A2 (mammaglobin 1); SCYEI (endothelial monocyte-activating cytokine); SDF2; SERPINA1; SERPINA3; SERP1NB5 (maspin); SERPINE1 (PAI -1); SERPDMF1; SHBG; SLA2; SLC2A2; SLC33A1; SLC43A1; SLIT2; SPPI; SPRR1B (Sprl); ST6GAL1; STABI; STAT6; STEAP; STEAP2; TB4R2; TBX21; TGFB2; TGFB3; TGFBI; TGFBRI; TGFBR2; TGFBR3; THIL; THBSI (thrombospondin-1); THBS2; THBS4; THPO; TIE (Tie-1); TMP3; Tissue factor; TLR1; TLR2 TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TLR10; TNF; TNF-a; TNFAEP2 (B94); ;TNFSF10 (TRAIL);TNFSF11 (TRANCE);TNFSF12 (AP03L);TNFSF13 (April);TNFSF13B;TNFSF14 (HVEM-L);TNFSF15 (VEGI);TNFSF18;TNFSF4 (OX40 ligand);TNFSF5 (CD40 ligand TNFSF6 (FasL); TNFSF7 (CD27 ligand); TNFSFS (CD30 ligand); TNFSF9 (4-1 BB ligand); TOLLIP; Toll-like receptors; TOP2A (topoisomerase Ea) TP53; TPM1; TPM2; TRADD; TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TREM1; TREM2; TRPC6; TSLP; TWEAK; VEGF; VEGFB; VEGFC; versican; VHL C5; VLA -4; XCL1 (lymphocyte chemotactic hormone); XCL2 (SCM-1b); XCRI (GPR5/CCXCRI); YY1;

Preferred molecular target molecules for antibodies produced using the methods provided herein (e.g., bispecific or multispecific antibodies) include CD proteins, such as the ErbB family of receptors, such as EGF receptors, HER2, HER3 or HER4 receptors Members of CD3, CD4, CDS, CD16, CD19, CD20, CD34, CD64, CD200; cell adhesion molecules, such as LFA-1, Mac1, p150.95, VLA-4, ICAM-1, VCAM, α4/β7 integrin and αv/β3 integrins, including their α or β subunits (eg, anti-CD11a, anti-CD18, or anti-CD11b antibodies); growth factors, such as VEGF-A, VEGF-C; tissue factor (TF); α-interferon (αIFN ); TNFα; interleukins such as IL-1β, IL-3, IL-4, IL-5, IL-S, IL-9, IL-13, IL 17AF, IL-1S, IL-13Rα1 , IL13R α2, IL-4R, IL-5R, IL-9R, IgE; blood group antigens; flk2/flt3 receptors; obesity (OB) receptors; mpl receptors; CTLA-4; RANKL, RANK, RSV F protein, Protein C, etc.

In one embodiment, an antibody (e.g., a bispecific or multispecific antibody) produced using the methods provided herein binds low-density lipoprotein receptor-related protein (LRP)-1 or LRP-8 or transferrin receptor , and at least one target selected from the group consisting of 1) beta-secretase (BACE1 or BACE2), 2) alpha-secretase, 3) gamma-secretase, 4) tau-secretase, 5) starch protein precursor protein (APP), 6) death receptor 6 (DR6), 7) amyloid beta peptide, 8) α-synuclein (alpha-synuclein), 9) Parkin (Parkin), 10 ) Huntingtin, 11) p75 NTR and 12) caspase-6.

In one embodiment, antibodies produced using the methods provided herein (e.g., bispecific or multispecific antibodies) bind to at least two target molecules selected from the group consisting of: IL-1α and IL-1 β, IL-12 and IL-1S; IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-5 and IL-4; IL-13 and IL-1β; IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and MEF; IL-13 and TGF-~; IL-13 and LHR agonist; IL-12 and TWEAK, IL -13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; IL-13 and ADAMS, IL-13 and PED2, IL17A and IL 17F, CD3 and CD19, CD138 and CD20; CD138 and CD40; CD19 and CD20; CD20 and CD3; CD3S and CD13S; CD3S and CD20; CD3S and CD40; CD40 and CD20; CD-S and IL-6; CD20 and BR3, TNF α and TGF-β, TNF α and IL-1 β; TNF α and IL-2, TNF α and IL-3, TNF α and IL-4, TNF α and IL-5, TNF α and IL6, TNF α and IL8, TNF α and IL-9, TNF α and IL-10, TNF α and IL-11, TNF α and IL-12, TNF α and IL-13, TNF α and IL-14, TNF α and IL-15, TNF α and IL-16, TNF α and IL-17, TNF α and IL-18, TNF α and IL-19, TNF α and IL-20, TNF α and IL-23, TNF α and IFN α, TNF α and CD4, TNF α and VEGF, TNF α and MIF, TNF α and ICAM-1, TNF α and PGE4, TNF α and PEG2, TNF α and RANK ligand, TNF α and Te38, TNF α and BAFF, TNF α and CD22, TNF α and CTLA-4, TNF α and GP130, TNF a and IL-12p40, VEGF and HER2, VEGF-A and HER2, VEGF-A and PDGF, HER1 and HER2, VEGFA and ANG2, VEGF-A and VEGF-C, VEGF-C and VEGF-D, HER2 and DR5, VEGF and IL-8, VEGF and MET, VEGFR and MET receptors, EGFR and MET, VEGFR and EGFR, HER2 and CD64, HER2 and CD3, HER2 and CD16, HER2 and HER3; EGFR (HER1) and HER2, EGFR and HER3 , EGFR and HER4, IL-14 and IL-13, IL-13 and CD40L, IL4 and CD40L, TNFR1 and IL-1 R, TNFR1 and IL-6R and TNFR1 and IL-18R, EpCAM and CD3, MAPG and CD28, EGFR and CD64, CSPGs and RGM A; CTLA-4 and BTN02; IGF1 and IGF2; IGF1/2 and Erb2B; MAG and RGM A; NgR and RGM A; NogoA and RGM A; OMGp and RGM A; POL-l and CTLA -4; and RGM A and RGM B.

Soluble antigens or fragments thereof, optionally conjugated to other molecules, can be used as immunogens for antibody production. For transmembrane molecules, such as receptors, fragments of these (eg, the extracellular domain of a receptor) can be used as immunogens. Alternatively, cells expressing transmembrane molecules can be used as immunogens. Such cells may be derived from natural sources such as cancer cell lines or may be cells that have been transformed by recombinant techniques to express transmembrane molecules. Other antigens and forms thereof suitable for use in preparing antibodies will be apparent to those skilled in the art. activity test

The physical/chemical properties and biological function of antibodies (eg, bispecific or multispecific antibodies) produced using the methods provided herein can be characterized by various assays known in the art. Such assays include, but are not limited to, N-terminal sequencing, amino acid analysis, native size exclusion high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography, and papain digestion.

In certain embodiments, antibodies (eg, bispecific or multispecific antibodies) produced using the methods provided herein are assayed for biological activity. In some embodiments, antibodies (eg, bispecific or multispecific antibodies) produced using the methods provided herein are tested for antigen binding activity. Antigen binding assays known in the art and useful herein include, but are not limited to, any direct or competitive binding assay using techniques such as western blot, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), " Sandwich" immunoassay, immunoprecipitation assay, fluorescent immunoassay and protein A immunoassay.

The foregoing written description is considered sufficient to enable those skilled in the art to practice the invention. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. Indeed various modifications in addition to those shown and described herein will be apparent from the foregoing description to those skilled in the art and are within the scope of the appended claims. Example Example 1 : Methods and Materials Antibody Construct Design and Synthesis

All antibodies in the following examples were prepared using Kabat (Kabat et al. "Sequences of Proteins of Immunological Interest." Bethesda, MD: NIH, 1991) and EU (Edelman et al. "The covalent structure of an entire gammaG immunoglobulin molecule." Proc Natl The Acad Sci USA 1969; 63:78-85) numbering system numbers the variable and constant domains separately. Antibody constructs were generated by gene synthesis (GENEWIZ®) as previously described (Dillon et al. "Efficient production of bispecific IgG of different isotypes and species of origin in single mammalian cells." MAbs 2017; 9:213-30) and Subcloning into expression plastids (pRK5) where applicable. All antibodies in this study were HC deglycosylated (N297G mutation) and the carboxy-terminal lysine (∆K447) was deleted to reduce product heterogeneity and thereby facilitate accurate quantification of BsIgG by LCMS (Dillon et al. (below); Yin et al. "Precise quantification of mixtures of bispecific IgG produced in single host cells by liquid chromatography-Orbitrap high-resolution mass spectrometry." MAbs 2016; 8:1467-76). The two-component HCs of all BsIgGs in this study were engineered to contain a 'knob' mutation in the first listed antibody (eg T366W) or a 'hole' mutation in the second listed antibody (eg T366S:L368A: Y407V) to promote HC heterodimerization (Atwell et al. "Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library." J Mol Biol 1997; 270:26-35).

For the few BsIgGs in this study, FR mutations were judiciously made to provide sufficient mass differences between correctly matched and mismatched BsIgG species for more accurate quantification by LCMS analysis. Accurate quantification of bispecific IgG yield requires a mass difference of ≤118 Da (Yin et al. (below)). Specifically, the antibodies and mutations were anti-HER2 V _L R66G when combined with anti-CD3 or variants (in Table A ), anti-IL-1β or anti-GFRα ( Table B ); when combined with anti-ANG2 or variants ( Table F middle) anti-VEGFA V _L F83A when combined; anti-CD3 V _L N34A:F83A when combined with anti-factor D 25D7 v1 or anti-IL-33 or anti-HER2 (in Table G2 ); anti-CD3 when combined RSPO3 V _L F83A; anti-EGFR V _L F83A when combined with anti-SIRPα or anti-Factor D 20D12 v1; and anti-IL-4 V _L N31A:F83A when combined with anti-GFRα1 ( Table B or Figures 1A-1F ) . The selected residues had no detectable effect on BsIgG yield based on comparison to the parental antibody. Antibody expression and purification

All BsIgG were transiently expressed in HEK293-derived EXPI293F™ cells as previously described (Dillon et al. (supra)). Four plasmids corresponding to two LCs and two HCs were co-transfected into EXPI293F™ cells (Thermo Fisher Scientific). The LC DNA was varied for each experiment and the highest bispecific yield was reported at the optimal HC:LC ratio as previously described (Dillon et al. (supra)). The ratio of the two HCs was fixed at 1:1. Transfected cell cultures (30 mL) were grown for 7 days at 37°C with shaking. BsIgG from filtered cell culture supernatants was purified in a high-throughput manner by Protein A affinity chromatography (TOYOPEARL® AF-rProtein A, Tosoh Bioscience). Impurities such as aggregates and half IgG ₁ were removed by size exclusion chromatography using a ZENIX®-C SEC-300 column (10 mm × 300 mm, 3 μm particle size, Sepax Technology). IgG ₁ concentrations were calculated using an extinction coefficient A ^0.1% _280nm of 1.5. Purification yield was estimated by multiplying protein concentration by elution volume after protein A chromatography. Characterization of BsIgG by SEC HPLC analysis

BsIgG was chromatographed by size exclusion chromatography under isocratic conditions on a TSKGEL® SuperSW3000 column (4.6 × 150 mm, 4 μm) (Tosoh Bioscience) connected to an HPLC column (DIONEX™ UltiMate 3000, Thermo Fisher Scientific) Sample (20 μL). The mobile phase was 200 mM potassium phosphate and 250 mM potassium chloride (pH 7.2), the flow rate was 0.3 mL/min, and the absorbance was measured at a wavelength of 280 nm. BsIgG yield determination by high-resolution LCMS

Quantification of BsIgG yield (intensity of correctly paired LC species versus all three mismatched IgG ₁ species) was performed via mass spectrometry (Thermo Fisher EXACTIVE™ Plus Extended Mass Range ORBITRAP™) as previously described, and between different mass peaks. No response bias was shown between (see Yin et al. (below)).

For denaturing mass spectrometry, samples (3 μg) were injected onto a reversed-phase liquid chromatography column (MABPAC™, Thermo Fisher Scientific, 2.1 mm × 50 mm). Binary gradient pumps were used to deliver solvent A (99.88% water containing 0.1% formic acid and 0.02% trifluoroacetic acid) and solvent B (90% acetonitrile containing 9.88% water plus 0.1% formic acid and 0.02% trifluoroacetic acid), Gradient of 20% to 65% solvent B at 300 μL/min over 4.5 min. The solvent was changed in steps over 0.1 min to 90% solvent B and held at 90% for 6.4 min to clean the column. Finally, the solvent was step changed to 20% solvent B over 0.1 min and held for 3.9 min for re-equilibration. Samples were analyzed via electrospray ionization to the midline of the mass spectrometer using the following parameters for data acquisition: 3.90 kV spray voltage; 325°C capillary temperature; 200 S-lens RF level; 15 sheath gas flow rate and 4 AUX in the ESI source Gas flow rate; 1,500 to 6,000 m/z scan range; desolvation, in-source CID 100 eV, CE 0; 17,500 resolution at m/z 200; positive polarity; 10 microscans; 3E6 AGC target; Fixed AGC mode; 0 averaging; 25 V source DC offset; 8 V injected flat pole DC; 7 V alternating planar lens; 6 V curved flat pole DC; 0 V transmit multipole DC tuning offset; Trap entrance lens tuning offset; and 2 for trapped gas pressure setting.

For native mass spectrometry, samples (10 μg) were injected onto an Acquity UPLC™ BEH size exclusion chromatography column (Waters, 4.6 mm × 150 mm) heated to 30°C using a Dionex ULTIMATE™ 3000 RSLC system. The isocratic chromatography operation (10 min) utilized an aqueous mobile phase containing 50 mM ammonium acetate (pH 7.0) at a flow rate of 300 μL/min.

Samples were analyzed via electrospray ionization to the midline of the mass spectrometer using the following parameters for data acquisition: 4.0 kV spray voltage; 320°C capillary temperature; 200 S-lens RF level; 4 sheath gas flow rates and 0 AUX in the ESI source Gas flow rate; 300 to 20,000 m/z scan range; desolvation, in-source CID 100 eV, CE 0; 17,500 resolution at m/z 200; positive polarity; 10 microscans; 1E6 AGC target; Fixed AGC mode; 0 averaging; 25 V source DC offset; 8 V injected flat pole DC; 7 V alternating planar lens; 6 V curved flat pole DC; 0 V transmit multipole DC tuning offset; Trap entrance lens tuning offset; and 2 for trapped gas pressure setting.

The acquired mass spectrometry data were analyzed using Protein Metrics Intact Mass™ software and Thermo Fisher BIOPHARMA FINDER™ 3.0 software. The signal intensities of the correctly paired LC species from the deconvoluted spectra of each sample were used for quantification relative to the three mismatched _IgGl species. HC homodimers and half IgG were not detectable or present in trace amounts and were excluded from calculations. Correctly LC-paired BsIgG was estimated from the isobaric mixture of BsIgG and double LC-mismatched IgG ₁ by using the algebraic formula described previously (see Yin et al. (below)). SDS-PAGE gel analysis of BsIgG

BsIgG purified by protein A and size exclusion chromatography was analyzed by SDS-PAGE. Samples were prepared for analysis of electrophoretic mobility under reducing and non-reducing conditions in the presence and absence of DTT, respectively. Samples mixed with sample dye were heated with DTT for 5 min or without DTT for 1 min at 95 °C and run on a 4-20% Tris-glycine gel (Bio-Rad) at 120 V. Electrophoresis. The gel was then stained with GELCODE ^™ blue protein stain (Thermo Fisher Scientific) and destained in water. An equal amount of protein (6 μg) was loaded for each sample. Kinetic Binding Experiment

Kinetic binding experiments were performed using surface plasmon resonance on a BIAcore T200 instrument (GE Healthcare). Anti-Fab (GE Healthcare) was immobilized [about 12000 resonance units (RU)] on the CM5 sensor wafer. Parental and mutant Fabs were captured onto immobilized surfaces and analyte binding assessed. Sensorgrams for the following analyte concentrations were generated at 25°C using an injection time of 3 minutes and a flow rate of 50 µl/min: 0, 0.293, 1.17, 4.6875, 18.75, 75, 300 nM HER2-ECD (in-house ) and VEGF-C (Cys156Ser) (R&D Systems, catalog number 752-VC); 0, 0.0195, 0.0781, 0.3125, 1.25, 5, 20 mM VEGF165 (R&D Systems, cat. Yes); 0, 0.0732, 0.293, 1.17, 4.6875, 18.75, 75 nM MET-R Fc (R&D Systems, Cat# 8614-MT), IL-1β (R&D Systems, Cat# 201-LB/CF), EGFR Fc (R&D Systems, Cat #344-ER); 0, 0.976, 3.906, 15.625, 62.5, 250 nM Biotinylated CD3 (proprietary). Dissociation was monitored 900 seconds after injection of the analyte. The working buffer used was 10 mM HEPES (pH 7.4), 150 mM NaCl, 0.003% EDTA, 0.05% Tween (HBS-EP+, GE Healthcare). The wafer surface was regenerated with 10 mM glycine (pH 2.1) after each injection. Sensorgrams were calibrated using a double blank reference (subtracting zero analyte concentration and a blank reference cell). The sensorgrams were then analyzed with the software provided by the manufacturer using the 1:1 Langmuir model. Example 2 : Elucidation of Heavy Chain / Light Chain Pairing Preference to Facilitate Assembly of Bispecific IgG in a Single Cell Introduction

In the studies described here, high-throughput production and high-resolution LCMS analysis (Dillon et al. "Efficient production of bispecific IgG of different isotypes and species of origin in single mammalian cells." MAbs 2017; 9:213-30 ; Yin et al. "Precise quantification of mixtures of bispecific IgG produced in single host cells by liquid chromatography-Orbitrap high-resolution mass spectrometry." MAbs 2016; 8:1467-76) was used to investigate 99 HCs with knobs and holes without Fab mutation BsIgG yields of different antibody pairs. One-third of the antibody pairs showed high (>65%) BsIgG yields, consistent with a strong cognate HC/LC chain pairing preference. For such antibody pairs a previously identified charge mutation was placed at the interface of the two _CH1 / _CL domains (Dillon et al. "Efficient production of bispecific IgG of different isotypes and species of origin in single mammalian cells." MAbs 2017 ; 9:213-30) for enhancing BsIgG production. Next, we investigated whether homologous strand pair preference in one or both arms was required for high yield of BsIgG. Mutation analysis was used to identify specific residues in CDR H3 and L3 that contributed to the high BsIgG yield. The identified CDR H3 and L3 and specific residues were then inserted into other available unrelated antibodies displaying random HC/LC chain pairing to determine their effect on BsIgG yield. Finally, mutation analysis was used to investigate the effect of interchain disulfide bonds on BsIgG yield. Effect of Constitutive Antibody on BsIgG Yield

Previously, high yields (>65% of BsIgG with knob heavy chain (HC) mutations but no Fab arm mutations) were observed for two bispecific antibodies, namely, anti-EGFR/MET and anti-IL-13/IL-4. ) (Dillon et al. (below)). To investigate the strength and frequency with which homologous heavy chain/light chain (HC/LC) pairing preference occurs, a large panel of antibody pairs (n = 99) was used to generate BsIgG. For simplicity, all bispecific antibodies in this study were constructed with human IgG ₁ HC constant domains. Six antibodies binding to IL-13, IL-4, MET, EGFR, HER2 or CD3 (Dillon et al. (below)) were used to construct a matrix of all 15 possible _BsIgG1s . These six antibodies were then arrayed with 14 additional antibodies, primarily of _the kappa LC isotype, three of which were of the lambda LC isotype (anti-DR5, anti- _α5β1 , anti-RSPO2) (see table below A ). In Table A , germline gene families were identified by comparing the LC and HC sequences to the human antibody germline gene repertoire using a dedicated alignment tool. The report has the closest match to the germline gene segment. Except for three fully human antibodies (anti-CD33, anti-PDGF-C, anti-Flu B), all antibodies used in this study were humanized antibodies. Table A : Germline gene family and LC isotype analysis of different antibodies evaluating LC/HC pairing preference. Antibody / clonal antigen binding specificity LC isotype germline gene family references V _L V _H Lerelizumab IL-13 kappa KV4 HV2 Ultsch et al. 19C11 IL-4 kappa KV1 HV3 Spiess et al. Onatuzumab/5D5 MET kappa KV1 HV3 Merchant et al. D1.5 EGFR kappa KV1 HV3 Schaefer et al. Trastuzumab/humAb4D5-8 HER2 kappa KV1 HV3 Carter et al. humAbUCHT1 v9 CD3 kappa KV1 HV3 Rodrigues et al. 25D7v1 Factor D kappa KV4 HV2 na 5D6 RSPO3 kappa KV1 HV4 na 10C12 IL-33 kappa KV3 HV3 na 19D1 v4.1 SIRPα kappa KV1 HV1 na 20D12v1 Factor D kappa KV1 HV1 na 8E11v2 LGR5 kappa KV4 HV1 na 2H12 v6.11 IL-1β kappa KV1 HV3 na 7C9 v8 GFRα1 kappa KV1 HV3 Bhakta et al. Apomab DR5 lambda LV3 HV3 Adams et al. 1A1 RSPO2 lambda LV2 HV3 na na α ₅ β ₁ lambda LV3 HV3 na 46B8 FluB kappa KV2 HV5 na 1E5 v3.1 PDGF-C kappa KV4 HV1 na GM15.33 CD33 kappa KV2 HV1 na KV = κ variable; LV = λ variable; HV = heavy variable; na = not available. 1. Merchant M, Ma X, Maun HR, Zheng Z, Peng J, Romero M, Huang A, Yang NY, Nishimura M, Greve J, et al. Monovalent antibody design and mechanism of action of onartuzumab, a MET antagonist with anti -tumor activity as a therapeutic agent. Proc Natl Acad Sci USA 2013; 110:E2987-96. 2. Schaefer G, Haber L, Crocker LM, Shia S, Shao L, Dowbenko D, Totpal K, Wong A, Lee CV, Stawicki S, et al. A two-in-one antibody against HER3 and EGFR has superior inhibitory activity compared with monospecific antibodies. Cancer Cell 2011; 20:472-86. 5. Ultsch M, Bevers J, Nakamura G, Vandlen R, Kelley RF, Wu LC, Eigenbrot C. Structural basis of signaling blockade by anti-IL-13 antibody lebrikizumab. J Mol Biol 2013; 425:1330-9. 6. Spiess C, Bevers J, 3rd, Jackman J, Chiang N, Nakamura G, Dillon M, Liu H, Molina P, Elliott JM, Shatz W, et al. Development of a human IgG4 bispecific antibody for dual targeting of interleukin-4 (IL-4) and interleukin-13 (IL-13) cytokines . J Biol Chem 2013; 288:26583-93. 8. Carter P, Presta L, Gorman CM, Ridgway JB, Henner D, Wong WL, Rowland AM, Kotts C, Carver ME, Shepard HM. Humanization of an anti-p185HER2 Antibody for human cancer therapy. Proc Natl Acad Sci USA 1992; 89:4285-9. 9. Rodrigues ML, Shalaby MR, Werther W, Presta L, Carter P. Engineering a humanized bispecific F(ab')2 fragment for improved binding to T cells. Int J Cancer Suppl 1992; 7:45-50. 10. Bhakta S, Crocker LM, Chen Y, Hazen M, Schutten MM, Li D, Kuijl C, Ohri R, Zhong F, Poon KA, et al . An anti-GDNF family receptor alpha 1 (GFRA1) antibody-drug conjugate for the treatment of hormone receptor-positive breast cancer. Mol Cancer Ther 2018; 17:638-49. 11. Adams C, Totpal K, Lawrence D, Marsters S, Pitti R, Yee S, Ross S, Deforge L, Koeppen H, Sagolla M, et al. Structural and functional analysis of the interaction between the agonistic monoclonal antibody Apomab and the proapoptotic receptor DR5. Cell Death Differ 2008; 15:751 -61.

Next, the antibody pairs shown in Table B below were co-expressed at optimized chain ratios in EXPI293F™ cells derived from HEK293, using a modified version of the previously described method (see Dillon et al., Yin et al. ( The yield of BsIgG was determined as follows)). None of the antibody pairs contained the Fab mutations described in Dillon et al. (below). All bispecific antibody pairs contained knob mutations for heavy chain heterodimerization.

Following antibody pair co-expression and protein A chromatography, the purified IgG ₁ pool was further purified by size exclusion chromatography (SEC) to remove any minor aggregates present prior to quantification by high-resolution LCMS and half IgG ₁ . The yield of correctly assembled BsIgG in an isobaric (ie, same molecular mass) mixture of IgG ₁ that also contained LC scrambled was estimated using a previously developed algebraic formula (see Yin et al. (below)). Data shown in Table B are the yields of BsIgG from optimized LC DNA ratios. BsIgG yields >65% are indicated in bold. The HC of mAb-1 contained a 'hole' mutation (T366S:S368A:Y407V) and the HC of mAb-2 contained a 'knob' mutation (T366W) (Atwell et al. "Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library.” J Mol Biol 1997; 270:26-35). Table B : Half-antibody pairs used to study BsIgG yield mAb-1 mAb-2 IL-13 MET EGFR CD3 IL-4 HER2 IL-13 NA 87.6 87.0 75.2 70.3 66.6 MET 86.6 NA 72.3 60.7 53.1 59.9 EGFR 86.3 72.4 NA 23.9 45.4 22.0 CD3 75.5 54.8 32.5 NA 25.0 22.7 IL-4 68.7 58.0 44.1 26.9 NA 22.6 HER2 64.6 65.4 21.6 24.1 25.0 NA DR5 90.4 95.1 53.3 53.4 53.8 34.7 FluB 87.7 69.5 52.3 32.0 60.8 72.7 RSPO3 84.7 58.6 82.1 40.6 26.0 22.0 Factor D 25D7 v1 83.6 73.1 69.3 83.1 35.5 68.7 RSPO2 83.5 51.1 78.5 38.7 22.3 71.3 IL-13 74.2 63.5 80.4 77.8 63.7 65.9 GFRα1 73.9 40.6 77.5 79.6 33.5 68.0 PDGF-C 61.2 71.0 54.6 56.0 34.2 24.3 CD33 49.8 58.8 49.6 36.4 56.5 51.5 α ₅ β ₁ 45.9 62.2 31.0 41.4 48.4 72.6 IL-33 45.6 21.4 30.9 20.4 42.4 46.6 SIRPα 41.7 31.0 22.6 60.6 47.9 31.8 Factor D 20D12 v1 23.5 29.8 58.0 36.0 22.6 69.6 LGR5 21.7 56.2 53.8 23.6 22.8 22.1 NA= not applicable; monospecific antibody.

The _BsIgG1 yields for the 99 unique antibody pairs varied over a very wide range: 22-95% ( see Table B ). Remarkably, non-random HC/LC pairing (>30% BsIgG ₁ yield) was observed for most (>80%) antibody pairs, with high (>65%) and Intermediate (30-65%) _BsIgG1 yield. Near quantitative (>90%) formation of _BsIgG1 was measured for two antibody pairs (anti-MET/DR5 and anti-IL-13/DR5).

Figures _1A -1F show low yields (<30%, for example anti-LGR5/IL-4, see Figures 1A and 1B ), intermediate yields (30%-65%, for example anti-SIRPα/IL-4, see Figures 1C and 1D ) and high-resolution LCMS data for representative examples of high yields (>65%, eg anti-MET/DR5, see Figures 1E and 1F ). The corresponding antibody pairs were transiently co-transfected into HEK293-derived EXPI293F™ cells. _IgGl species were purified by protein A chromatography and size exclusion chromatography prior to quantification of _BsIgGl yield by high-resolution LCMS as described in Dillon et al. (below) and Yin et al. (below). The data shown in Figures 1A , 1C and 1E are the mass envelopes for charge states 38+ and 39+, and Figures 1B , 1D and 1F show the corresponding deconvoluted data and provide animations representative of the different IgG ₁ species present.

The _BsIgG1 yield of each antibody studied varied widely depending on its partner antibody. For example, _BsIgG1 yields for anti-MET antibodies varied from as low as about 21% when paired with anti-IL-33 to as high as about 95% when paired with anti-DR5 ( Table B ). To investigate any effect of the 'knob' and 'hole' mutations on the pairing preference of cognate HC/LC, _BsIgG1 were produced from HCs containing the 'knob' mutation in mAb1 and the 'hole' mutation in mAb2, and vice versa ( Table B ). The yield of BsIgG ₁ was minimally affected by HC containing "knob" and "hole" mutations in all cases tested (n=15) ( Table B ). Recovery of IgG species from 30 mL cultures by protein A chromatography varied by more than about 5-fold (1.5 to 8.0 mg).

The above results indicate that the high yield of _BsIgG1 without Fab mutations is a common phenomenon dependent on constitutive antibody pairs. Effect of _CH 1/ _CL Interface Charge Mutations on BsIgG1 Yield of Antibody Pairs with Homologous HC/LC Pairing Preference

Previously, a combination of mutations at all four domain/domain interfaces (i.e., two _VH / _VL and two _CH1 / _CL ) in combination with knob-and-hole HC mutations was used in a single mammalian host cell Near-quantitative assembly of BsIgG of different isotypes in (see Dillon et al. (below)). Here, antibody pairs that produced high yields of _BsIgG1 without any Fab mutation were identified ( Table B ). These antibody pairs differ in their variable domain sequences, while the constant domains, ie, IgG _{1 CH} 1 and k _CL , are in most cases identical. It was hypothesized that for such antibody pairs, mutations at the two _CH1 / _CL interfaces alone might be sufficient to increase the yield of correctly assembled bispecific antibodies to approximately 100%. Eleven different antibody pairs were selected and the yields of _BsIgG1 were compared in the presence or absence of previously reported _CH1 / _CL domain interface charge mutations (see Dillon et al. (below)). Specifically, the "knob" arm was engineered to have the _CL V133E and CH ₁ S183K mutations and the "hole" arm had the _CL V133K and CH ₁ S183E mutations (see Dillon et al. (below)). Charge mutations at the two _CH1 / _CL interfaces increased _BsIgG1 yields for all antibody pairs by about 12-34% in most (9/11) cases to ≥ 90% _BsIgG1 yields ( Figure 2 ). For the charge pair variants in Figure 2 , the first listed antibody in the pair contains the _CL V133E and CH ₁ S183K mutations, and the second listed antibody contains the _CL V133K and CH ₁ S183E mutations (see Dillon et al. people (below)). The 90% yield of _BsIgG1 is indicated by the horizontal dashed line in FIG. 2 . The _CLV133E and _CH1S183K mutations did not affect the affinity of the antibody for its target antigen (data not shown). Effect of homologous HC/LC pairing preference in one arm of BsIgG on BsIgG yield

The mechanistic basis for the high yield of _BsIgG1 observed for some antibody pairs was investigated. Two antibody pairs, namely, anti-EGFR/MET and anti-IL-4/IL-13 were selected in this study based on the high yield of _BsIgG1 without Fab mutations ( see Table B and Dillon et al. (below)). A priori, one or both Fabs may exhibit a homologous HC/LC pairing preference that contributes to the high yield of _BsIgG1 . Three chain co-expression experiments were employed to discriminate between these possibilities. A single HC (HC1) with a 'knob' or 'hole' mutation was transiently co-expressed in Expi293F™ cells with its cognate LC (LC1) and a competing non-cognate LC (LC2) ( Figure 3 ). Asterisks in Figure 3 indicate the presence of "knob" or "hole" mutations in HC. (Anti-EGFR, anti-IL13, and anti-HER2 HCs contain a "knob" mutation (T366W), while anti-MET, anti-IL4, and anti-CD3 HCs contain a "hole" mutation (T366S:S368A:Y407V) (see Atwell et al., "Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library." J Mol Biol 1997; 270:26-35).) The resulting half IgG species were purified from the corresponding cell culture supernatants by protein A affinity chromatography And the extent of homologous and non-homologous HC/LC pairing was assessed by high-resolution LCMS (Dillon et al. and Yin et al. (below)). The percentage of homologous HC/LC pairings was calculated by quantifying _half IgGl species.

As shown in Table C below, anti-MET HCs showed a stronger preference for their cognate LCs (about 71%) than non-cognate anti-EGFR LCs, while anti-EGFR HCs showed a preference for their cognate LCs (about 56%) only Slightly more than non-cognate anti-MET LC. Anti-IL-13 HCs showed a stronger preference for their cognate LCs (81%) than non-cognate anti-IL-4 LCs, whereas anti-IL-4 HCs showed no preference for their cognate LCs (49%). These data are consistent with the notion that the high _BsIgG1 yield of anti-EGFR/MET is caused by strong and weak cognate HC/LC pairing preferences of anti-MET and anti-EGFR antibodies, respectively. In contrast, the high _BsIgG1 yield of anti-IL-13/IL-4 apparently reflects the strong cognate HC/LC pairing preference of the anti-IL-13 antibody alone. Thus, homologous HC/LC pairing preferences in one or both arms may apparently be sufficient for high yields of _BsIgG1 in single cells without Fab mutations. Table C : Quantification of antibody homologous chain bias after co-expression. HC1 LC1 LC2 HC/LC pairing (%) Homologous non-homologous MET MET EGFR 70.6 29.4 EGFR MET EGFR 56.4 43.6 IL-13 IL-13 IL-4 81.0 19.0 IL-4 IL-13 IL-4 49.1 50.9 HER2 HER2 CD3 51.0 49.0 CD3 HER2 CD3 46.4 53.6

Anti-HER2/CD3 was chosen as a control for this study based on its low yield of _BsIgG1 ( see Table B and Dillon et al. (below)). Anti-HER2 HCs showed no pairing preference for their cognate LCs over non-cognate anti-CD3 LCs. Similarly, anti-CD3 HCs showed no pairing preference for their cognate LCs over non-cognate anti-HER2 LCs ( see Table C ).

Pairing of HC with its cognate light chain (LC) or non-cognate LC when co-expressed in a single host cell was also evaluated. Briefly, each HC was co-transfected into HEK293-derived EXPI293F™ cells together with its cognate LC or non-cognate LC. IgG1 and half IgG1 species were purified from cell culture supernatants by protein A chromatography and analyzed by LC-MS. (Labrijn et al. "Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange." Proc Natl Acad Sci USA 2013; 110:5145-50; Spiess C et al. "Bispecific antibodies with natural architecture produced by co-culture of bacteria expressing two distinct half-antibodies.” Nat Biotechnol 2013; 31:753-8). The percentage of homologous HC/LC pairings was calculated by quantifying half IgGl species. Protein expression yields were estimated by multiplying the antibody concentration by the elution volume obtained from the high-throughput protein A chromatography step. Anti-EGFR, anti-IL-13, and anti-HER2 HCs contain a "knob" mutation (T366W), while anti-MET, anti-IL-4, and anti-CD3 HCs contain a "hole" mutation (T366S:S368A:Y407V) (see Spiess et al. "Alternative molecular formats and therapeutic applications for bispecific antibodies." Mol Immunol 2015; 67:95-106). In the absence of competition, HCs could be efficiently assembled with non-cognate LCs as judged by all six different mismatched HC/LC pairs tested ( see Table D below). Table D : Pairing of HCs with their cognate light chain (LC) or non-cognate LC when co-expressed in a single host cell HC LC half IgG ₁ Expressed Yield (mg) HC-LC pairing (%) MET MET 100.0 6.3 MET EGFR 100.0 6.7 EGFR EGFR 100.0 5.1 EGFR MET 100.0 6.6 IL-13 IL-13 100.0 3.0 IL-13 IL-4 100 1.9 IL-4 IL-4 100 4.8 IL-4 IL-13 100 3.1 HER2 HER2 100 5.4 HER2 CD3 100 6.1 CD3 CD3 100 4.1 CD3 HER2 100 5.0 Contribution of anti- MET CDR L3 and CDR H3 to the yield of anti- EGFR/MET BsIgG ₁

Sequence determinants in anti-MET antibodies that contribute to the high bispecific yield of anti-EGFR/MET BsIgG ₁ were investigated. The amino acid sequence differences between the anti-EGFR and anti-MET antibodies are all within the CDRs, plus an additional framework region (FR) residue, _VH94 , immediately adjacent to CDR H3 ( Figure 4 ). The remaining FR plus _Ck and _CH1 constant domain sequences of these antibodies were identical ( Figure 4 ). As evidenced by the X-ray crystal structure of the anti-MET Fab in complex with its antigen (Protein Data Bank (PDB) identifier code 4K3J) (see Merchant et al. "Monovalent antibody design and mechanism of action of onartuzumab, a MET antagonist with anti-tumor activity as a therapeutic agent.” Proc Natl Acad Sci USA 2013; 110:E2987-96), CDR L3 and H3 are the most widely involved CDRs at the _VH / _VL domain interface of anti-MET antibodies. These observations led to the hypothesis that CDR L3 and H3 of anti-MET antibodies may contribute to the high bispecific yield of anti-EGFR/MET BsIgG ₁ . Consistent with this notion, substitution of both CDR L3 and H3 of the anti-MET antibody by the corresponding sequences from the anti-CD3 antibody resulted in a substantial loss of bispecific yield (approximately 85% to 33%, FIG. 5A ). In contrast, replacing both CDR L3 and H3 of the anti-EGFR arm of the anti-EGFR/MET bispecific antibody caused only a small decrease in BsIgG yield (approximately 85% to 75%, FIG. 5A ). Substitution of CDR L3 and H3 of both anti-EGFR and anti-MET arms resulted in random HC/LC pairing. These data support the notion that anti-MET CDR L3 and H3 make a major contribution to the high bispecific yields observed for anti-EGFR/MET BsIgG ₁ , while anti-EGFR CDR L3 and H3 make a minor contribution. Substitution of CDR L1 and H1 or CDR L2 and H2 from the anti-MET antibody by the corresponding anti-CD3 antibody sequence had little to no effect on the bispecific yield of anti-EGFR/MET BsIgG ( FIG. 6 ). Contribution of residues within anti -METCDR L3 and CDR H3 to the yield of anti- EGFR/MET BsIgG ₁

Next, residues within CDR L3 and H3 of the anti-MET antibody that contribute to the high bispecific yield of anti-EGFR/MET BsIgG ₁ were investigated. The X-ray crystal structure of the anti-MET Fab (PDB accession 4K3J) revealed the contact residues between CDR L3 and H3 ( Figure 7 ) and was used to guide the selection of residues for mutation analysis. Alanine-scanning mutagenesis of anti-MET CDR L3 and H3 (Cunningham et al. "High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis." Science 1989; 244:1081-5) for mapping against EGFR/MET Residues Contributing to High Bispecific Yield of BsIgG ₁ . Table E1 : Alanine Scanning Mutagenesis of CDR L3 and H3 Contact Residues of Anti- MET Antibodies Anti- EGFR/MET BsIgG ₁ Anti- MET variant Yield (%) CDR L3 CDR H3 Parents Parents 83.6 ± 3.5 Y91A Parents 57.3±1.0 Y92A Parents 89.5±0.2 Y94A Parents 68.2 ± 4.9 P95A Parents 85.8±1.0 W96A Parents 70.1±0.9 Y91A:Y94A Parents 22.6±0.4 Y91A:W96A Parents 35.1 ± 1.7 Y94A:W96A Parents 56.0 ± 0.2 Y91A:Y94A:W96A Parents 23.2±0.2 Parents Y95A 74.9±0.9 Parents R96A 78.3 ± 2.8 Parents S97A 82.7 ± 3.9 Parents Y98A 79.0 ± 0.1 Parents V99A 79.8±0.9 Parents T100A 85.5±0.7 Parents P100Aa 64.7±4.7 Parents V99A:P100aA 72.8±4.2

As shown in Table E1 above, the _VLY91A mutation in CDR L3 caused the greatest reduction in bispecific yield (84% to 57%) of any of the 12 single alanine mutants tested. As few as two alanine substitutions in CDR L3, ie, VL _Y91A :Y94A, abolished high bispecific yields (84% to 23%). Thus, CDR L3 residues V _L Y91 and Y94 appear to be critical contributors to the high bispecific yield of anti-EGFR/MET BsIgG ₁ . All mutants exhibited comparable potency to the parental _BsIgG1 as estimated from yields recovered from protein A chromatography (data not shown). Data shown in Table E1 represent ± standard deviation of two independent experiments using optimized HC/LC DNA ratios ( see Table B ).

The affinity for MET of the parental anti-MET Fab and a subset of anti-MET Fab variants in Table E1 was determined via surface plasmon resonance (SPR). Association rates (k _on ), dissociation rates (k _off ) and binding affinities (K _D ) are shown in Table E2 ( nd indicates no detection of binding). The P95A substitution in CDR L3 did not affect the binding of anti-MET Fab variants to MET. Other single alanine substitutions in CDR L3 reduced affinity to varying degrees. No binding to antigen was detected for anti-Met Fab variants with Y91A:Y94A or Y91A:W96A double substitutions in CDR L3. Table E2 Parental anti -MET Fab and Fab variants CDR L3 CDR H3 k _on (x 10 ⁴ M ^-1 s ^-1 ) k _off (x 10 ^-4 s ^-1 ) K _D (nM) Parents Parents 17.9 < 0.1 ＜ 0.05 Y91A Parents 7.0 0.6 0.8 Y92A Parents 17.2 1.9 1.1 Y94A Parents 11.5 6.5 5.7 P95A Parents 15.3 < 0.1 ＜ 0.06 W96A Parents 8.4 1.7 2.1 Y91A:Y94A Parents nd nd nd Y91A:W96A Parents nd nd nd Contribution of anti -IL13 CDR L3 and CDR H3 to the yield of anti -IL13/IL14 BsIgG ₁

Given the discovery that specific residues in CDR L3 of anti-MET antibodies are important for the high bispecific yield of anti-EGFR/MET BsIgG ₁ , it was postulated that a similar principle could be applied for high bispecific yield of anti-IL-13/IL-4 BsIgG ₁ Anti-IL-13 antibodies contributed to the specific yield. A similar experimental strategy was used to investigate this possibility. A striking difference between these two antibody pairs is that both the CDR and FR sequences of the anti-IL-13 and anti-IL-4 antibodies are different ( Figure 8 ), while the anti-MET and anti-EGFR antibodies have identical FR sequences (except for V _H 94) and their CDR sequences are different ( Figure 4 ).

Replacement of CDR L3 and H3 of the anti-IL-13 antibody with the corresponding sequences from the anti-CD3 antibody resulted in a substantial loss of bispecific yield of anti-IL-13/IL-4 BsIgG ₁ (approximately 72% to 37%, Figure 5B ). In contrast, a slight increase was observed when CDR L3 and H3 of the anti-IL-4 antibody were replaced in a similar manner ( FIG. 5B ). These results indicate that CDR L3 and H3 of the anti-IL-13 antibody contribute to the high bispecific yield of anti-IL-13/IL-4 BsIgG ₁ .

Alanine scanning mutation analysis of anti-IL-13 CDR L3 and H3 (Cunningham et al. (infra)) was used to map residues that contribute to the high bispecific yield of anti-IL-13/IL-4 BsIgG ₁ . X-ray crystal structure of anti-IL-13 Fab in complex with IL-13 (PDB accession 4I77, see Ultsch et al. "Structural basis of signaling blockade by anti-IL-13 antibody lebrikizumab." J Mol Biol 2013; 425:1330 -9) Exposure of contact residues between CDR L3 and H3 ( FIG. 9 ) and used to select residues for mutation analysis ( Table F1 below). CDR L3 mutation _VL R96A caused the greatest reduction in bispecific yield of any of the nine single alanine mutants tested for CDR L3 and H3 and eliminated the high bispecific yield (72% to 29%) ). As few as two alanine substitutions in CDR H3, ie, _VH D95A:P99A, also abolished high bispecific yields (72% to 26%). All mutants exhibited comparable potency to the parental _BsIgG1 as estimated from yields recovered from protein A chromatography (data not shown). Data shown in Table F1 represent ± standard deviation of two independent experiments using optimized HC/LC DNA ratios ( see Table B ). Table F1 : Alanine Scanning Mutagenesis of CDR L3 and H3 Contact Residues of Anti -IL13 Antibodies Anti- IL-13/IL-4 BsIgG ₁ Anti- IL13 variant Yield (%) CDR L3 CDR H3 Parents Parents 71.8 ± 1.6 N91A Parents 65.4±2.1 N92A Parents 69.7±1.1 D94A Parents 78.1 ± 3.3 R96A Parents 28.7±1.4 N91A:D94A Parents 68.7 ± 3.5 D94A:R96A Parents 24.8±2.1 N91A:D94A:R96A Parents 36.8±0.1 Parents D95A 55.9 ± 0.1 Parents Y97A 77.0 ± 1.9 Parents Y98A 63.7±0.7 Parents P99A 72.5±1.3 Parents Y100A 55.7 ± 2.8 Parents D95A:P99A 26.1 ± 2.9

Thus, CDR L3 and/or H3 may make a critical contribution to the high bispecific yield as judged by both anti-EGFR/MET and anti-IL-13/IL-4 BsIgG ₁ studied here.

The affinity for IL-13 of the parental anti-IL-13 Fab and subset of anti-IL-13 Fab variants in Table F1 was determined by SPR. Association rates (k _on ), dissociation rates (k _off ) and binding affinities (K _D ) are shown in Table F2 ( nd indicates no detection of binding). Neither the N92A nor the D94A substitution in CDR L3 affected the binding of the anti-IL-13 Fab variants to IL-13. The R96A substitution in CDR L3 resulted in an approximately 10-fold loss of binding affinity, as did the D94:R96A double substitution in CDR L3. Other single alanine substitutions in CDR H3 reduced affinity to varying degrees. No binding to antigen was detected for the D95A:P99A double substitution in CDR H3. Table E2. Parental anti-IL-13 Fab and Fab variants CDR L3 CDR H3 k _on (x 10 ⁴ M ^-1 s ^-1 ) k _off (x 10 ^-4 s ^-1 ) K _D (nM) Parents Parents 117.1 0.5 0.05 N92A Parents 103.0 0.3 0.03 D94A Parents 124.3 0.3 0.02 R96A Parents 82.5 4.4 0.5 D94A:R96A Parents 52.8 3.7 0.7 Parents D95A 88.2 11.1 1.3 Parents P99A 150.4 26.9 1.8 Parents D95A:P99A nd nd nd The effect of CDR L3 and CDR H3 on the yield of BsIgG ₁

Next, a series of experiments were performed to determine whether CDR L3 and H3 from these antibodies could be sufficient to provide high bispecific yields for other antibody pairs. Two antibody pairs with low bispecific yields were selected, i.e., anti-HER2/CD3 (22-24%) and anti-VEGFA/ANG2 (24%) ( see Table B and Dillon et al. (below)), and CDR L3 and H3 of one arm of each of these two BsIgG _1s were replaced with the corresponding CDR sequences from anti-MET or anti-IL-13 antibodies. For both anti-HER2/CD3 ( FIG. 10A ) and anti-VEGFA/ANG2 ( FIG. 10B ), substantial increases in _BsIgG1 yield (from about 24% were observed in three of the four CDR L3 and H3 recruitment scenarios up to 40-65%). Data presented in Figures 10A and 10B are from optimized LC DNA ratios. The data in Figures 10A and 10B indicate that recruitment of CDR L3 and H3 from antibodies with cognate HC/LC pairing preference can increase the yield of _BsIgG1 without pairing preference, but not always.

The effect of recruitment of single key residues from anti-IL-13 antibodies to other antibodies on BsIgG1 yield was investigated. See Table G1 below. Amino acid numbering is according to Kabat. Antibodies containing variable domain mutations are indicated in bold. Data shown are from optimized LC DNA ratios. Anti-VEGFC with an aspartic acid residue at position 95 (D95) was not mutated. Table G1 : Recruitment of single key residues from anti -IL13 antibodies to other antibodies to study the effect on BsIgG ₁ yield BsIgG ₁ CDR L3 CDR H3 BsIgG ₁ yield (%) Anti-HER2/CD3 Parents Parents 24.0 Anti- HER2 /CD3 T94D Parents 47.5 Anti- HER2 /CD3 P96R Parents 40.1 Anti- HER2 /CD3 Parents W95D 36.0 anti-VEGFA/ANG2 Parents Parents 22.1 Anti- VEGFA /ANG2 V94D Parents 23.8 Anti- VEGFA /ANG2 W96R Parents 23.5 Anti- VEGFA /ANG2 Parents Y95D 22.7 anti-VEGFC/CD3 Parents Parents 24.1 anti- VEGFC /CD3 T94D Parent (D95) 44.0 anti- VEGFC /CD3 P96R Parent (D95) 31.7

Some increase in bispecific yield was observed when two or more key residues of anti-IL-13's pairing preference were grafted into corresponding positions in anti-HER2, anti-VEGFA or anti-VEGFC antibodies, but less than Parental anti-IL-13/IL-4 BsIgG ₁ (see Table G2 below ). In Table G2 , antibodies containing variable domain mutations are indicated in bold and amino acid numbering is according to Kabat. Antibodies containing variable domain mutations are in bold and underlined text. Data shown represent mean ± SD of two independent experiments using optimized LC DNA ratios. Anti-VEGFC with an aspartic acid residue at position 95 (D95) was not mutated. Table G2 : Recruitment of key amine groups from anti -IL13 antibodies to other antibodies to study impact on BsIgG ₁ yield BsIgG ₁ CDR L3 CDR H3 BsIgG ₁ yield (%) Anti-HER2/CD3 Parents Parents 24.0 Anti- HER2 /CD3 T94D:P96R Parents 31.8 Anti- HER2 /CD3 Parents W95D 36.0 Anti- HER2 /CD3 T94D:P96R W95D 47.4 anti-VEGFA/ANG2 Parents Parents 22.1 Anti- VEGFA /ANG2 V94D:W96R Parents 52.5 Anti- VEGFA /ANG2 Parents Y95D 22.7 Anti- VEGFA /ANG2 V94D:W96R Y95D 59.1 anti-VEGFC/CD3 Parents Parent (D95) 24.1 anti- VEGFC /CD3 T94D:P96R Parent (D95) 50.4

Taken together, these results suggest that charged residues at positions 94 and 96 (Kabat numbering) of CDR L3 and at position 95 (Kabat numbering) of CDR H3 (such as D and R) can affect the pairing of some but not all antibody pairs preference.

The affinity of the parental anti-HER2, anti-VEGFA and anti-VEGFC Fabs and subsets of anti-HER2, anti-VEGFA and anti-VEGFC Fab variants in Tables G1 and G2 to their respective targets was determined by SPR. Association rates (k _on ), dissociation rates (k _off ) and binding affinities (K _D ) are shown in Table G3 ( nd indicates no binding detected). Transfer of critical residues from anti-IL13 to other antibodies resulted in loss of binding affinity. Remarkably, the T94D substitution in CDR-L3 of anti-HER2 increased the _BsIgG1 yield of anti-HER2/anti-CD3 BsAbs from 24% to about 50%, while only reducing the affinity of anti-HER2 for HER2 by 20-fold. Similarly, the V94D:W96R double substitution in CDR-L3 of VEGFA increased the _BsIgG1 yield of anti-VEGFA/anti-ANG2 BsAbs from about 22% to about 52%, while only reducing the affinity of anti-VEGFA for VEGFA by about 20-fold . Table G3 fab CDR L3 CDR H3 k _on (x 10 ⁴ M ^-1 s ¹ ) k _off (x 10 ^-4 s ¹ ) K _D (nM) Anti-HER2 Parents Parents 10.4 1.3 1.2 T94D Parents 6.9 16.8 24.4 P96R Parents 7.0 149.5 212.9 Parents W95D 8.0 29.4 36.5 T94D:P96R Parents nd nd nd T94D:P96R W95D nd nd nd anti-VEGFA Parents Parents 65.4 < 0.1 ＜ 0.015 V94D Parents 59.8 < 0.1 ＜ 0.016 W96R Parents 13.3 9.1 6.8 Parents Y95D 92.6 6.0 0.6 V94D:W96R Parents 163.8 4.7 0.3 T94D:P96R W95D nd nd nd anti-VEGFC Parents Parents 17.1 14.1 8.2 V94D Parents nd nd nd W96R Parents nd nd nd Parents Y95D nd nd nd

In contrast to the results shown in Tables G1 and G2 , when residues key to the pairing preference of anti-cMet were grafted to the corresponding positions in the anti-HER2, anti-VEGFA or anti-VEGFC antibodies, doublets were observed in most cases. Small increase in specific yield. See Table G4 below. In Table G4 , antibodies containing variable domain mutations are indicated in bold and amino acid numbering is according to Kabat. Table G4 : Recruitment of key amine groups from anti- cMet antibodies to other antibodies to study impact on BsIgG ₁ yield BsIgG ₁ CDR L3 CDR H3 BsIgG ₁ yield (%) Anti-HER2/CD3 Parents Parents 24.0 Anti- HER2 /CD3 H91Y Parents 23.6 Anti- HER2 /CD3 T94Y Parents 31.0 Anti- HER2 /CD3 P96W Parents 26.2 Anti- HER2 /CD3 H91Y:T94Y Parents 24.2 Anti- HER2 /CD3 H91Y:P96W Parents 23.4 Anti- HER2 /CD3 T94Y:P96W Parents 22.7 Anti- HER2 /CD3 H91Y:T94Y:P96W Parents 23.6 anti-VEGFA/ANG2 Parent (Y91, W96) Parents 22.1 Anti- VEGFA /ANG2 (Y91)V94Y(W96) Parents 23.6 anti-VEGFC/CD3 Parents Parents 23.9 anti- VEGFC /CD3 S91Y Parents 22.6 anti- VEGFC /CD3 T94Y Parents 33.6 anti- VEGFC /CD3 P96W Parents 47.7 anti- VEGFC /CD3 S91Y:T94Y Parents 22.4 anti- VEGFC /CD3 S91Y:P96W Parents 59.0 anti- VEGFC /CD3 T94Y:P96W Parents 36.4 anti- VEGFC /CD3 S91Y:T94Y:P96W Parents 47.8 Anti-HER2/EGFR Parents Parents 21.4 Anti- HER2 /EGFR H91Y:T94Y Parents 22.3 Anti- HER2 /EGFR H91Y:P96W Parents 24.2 Anti- HER2 /EGFR T94Y:P96W Parents 23.4 Anti- HER2 /EGFR H91Y:T94Y:P96W Parents 33.6 Contribution of interchain disulfide bonds to BsIgG ₁ yield

Previously, it was hypothesized that interchain disulfide bond formation between HC and LC acts as a kinetic trap preventing strand exchange (Dillon et al. (below)). Experiments were performed to investigate whether the disulfide bond between HC and LC affects two BsIgG ₁ (anti-EGFR/MET and anti-IL-13/IL-4) with a clear homologous strand preference and two BsIgG 1 with a random HC/LC pairing. Bispecific yields of two controls (anti-HER2/CD3 and anti-VEGFA/VEGFC). Briefly, cysteine to serine mutations: LC C214S and HC C220S were used to generate _BsIgG1 variants lacking interchain disulfide bonds. Removal of interchain disulfide bonds in engineered variants was verified by SDS PAGE. As indicated in Figure 11 , samples were subjected to electrophoresis under reducing or non-reducing conditions. Four different BsIgG1 were analyzed: anti-HER2/CD3 (lane 1); anti-VEGFA/VEGFC (lane 2); anti-EGFR/MET (lane 3); and anti-IL13/IL14 (lane 4). As shown in Table H below, no clear evidence was found that interchain disulfide bonds affected the yield of BsIgG ₁ for any of the four antibody pairs tested, as judged by native mass spectrometry. _BsIgG1 yields were similar for the parental and disulfide engineered variants. Data in Table H are the mean ± standard deviation of three biological replicates using optimized DNA light chain ratios. Table H : Mutation analysis to determine the effect of the disulfide bond between HC and LC on BsIgG ₁ yield. BsIgG ₁ yield (%) BsIgG ₁ Parents with HC/LC disulfide bonds Variants without HC/LC disulfide bonds Anti-EGFR/MET 81.1 ± 1.4 82.8 ± 2.6 Anti-IL-13/IL-4 73.3±4.5 75.1±0.8 Anti-HER2/CD3 24.5±0.8 27.0 ± 2.4 Anti-VEGFA/VEGFC 28.8 ± 5.9 38.0 ± 6.0

In conclusion, this study demonstrates that cognate HC/LC pairing preference for BsIgG production in single cells is a common phenomenon that is strongly dependent on specific antibody pairs. Mechanistically, this strand pairing preference can be strongly influenced by residues in CDR H3 and L3. Indeed, this pairing preference can be exploited to reduce the number of Fab mutations used to drive the production of BsIgG ₁ and potentially other isotypes of BsIgG in a single cell. Additional references Brinkmann U, Kontermann RE. The making of bispecific antibodies. mAbs 2017; 9:182-212. Carter PJ, Lazar GA. Next generation antibody drugs: pursuit of the 'high-hanging fruit'. Nat Rev Drug Discov 2018 ; 17:197-223. Sanford M. Blinatumomab: first global approval. Drugs 2015; 75:321-7. Oldenburg J, Mahlangu JN, Kim B, Schmitt C, Callaghan MU, Young G, Santagostino E, Kruse-Jarres R , Negrir C, Kessler C, et al. Emicizumab prophylaxis in hemophilia A with inhibitors. N Engl J Med 2017; 377:809-18. Scott LJ, Kim ES. Emicizumab-kxwh: First Global Approval. Drugs 2018; 78:269 -74. Suresh MR, Cuello AC, Milstein C. Bispecific monoclonal antibodies from hybrid hybridomas. Methods Enzymol 1986; 121:210-28. Fischer N, Elson G, Magistrelli G, Dheilly E, Fouque N, Laurendon A, Gueneau F, Ravn U, Depoisier JF, Moine V, et al. Exploiting light chains for the scalable generation and platform purification of native human bispecific IgG. Nat Commun 2015; 6:6113. Strop P, Ho WH, Boustany LM, Abdiche YN, Lindquist KC , Farias SE, Rickert M, Appah CT, Pascua E, Radcliffe T, et al. Generating bispecific human IgG1 and IgG2 antibodies from any antibody pair. J Mol Biol 2012; 420:204-19. Vaks L, Litvak-Greenfeld D, Dror S, Matatov G, Nahary L, Shapira S, Hakim R, Alroy I, Benhar I. Design principles for bispecific IgGs, opportunities and pitfalls of artificial disulfide bonds. Antibodies 2018; 7. Schaefer W, Völger HR, Lorenz S, Imhof -Jung S, Regula JT, Klein C, Mølhøj M. Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry. MAbs 2016; 8:49-55. Bönisch M, Sellmann C, Maresch D, Halbig C, Becker S, Toleikis L, Hock B, Ruker F. Novel CH1:CL interfaces that enhance correct light chain pairing in heterodimeric bispecific antibodies. Protein Eng Des Sel 2017; 30:685-96. Kitazawa T, Igawa T, Sampei Z, Muto A, Kojima T, Soeda T, Yoshihashi K, Okuyama-Nishida Y, Saito H, Tsunoda H, et al. A bispecific antibody to factors IXa and X restores factor VIII hemostatic activity in a hemophilia A model. Nature Medicine 2012; 18:1570-4. Sampei Z, Igawa T, Soeda T, Funaki M, Yoshihashi K, Kitazawa T, Muto A, Kojima T, Nakamura S, Hattori K. Non-antigen-contacting region of an asymmetric bispecific antibody to factors IXa/X significantly affects factor VIII-mimetic activity. MAbs 2015; 7:120-8. Sampei Z, Igawa T, Soeda T, Okuyama-Nishida Y, Moriyama C, Wakabayashi T, Tanaka E, Muto A, Kojima T, Kitazawa T, et al. Identification and multidimensional optimization of an asymmetric bispecific IgG antibody mimicking the function of factor VIII cofactor activity. PLoS One 2013; 8:e57479. Carter PJ. Introduction to the current and future apeut proteins: protein engineering perspective. Exp Cell Res 2011. Wu H, Pfarr DS, Johnson S, Brewah YA, Woods RM, Patel NK, White WI, Young JF, Kiener PA. Development of motavizumab, an ultra-potent antibody for the prevention of respiratory syncytial virus infection in the upper and lower respiratory tract. J Mol Biol 2007; 368:652-65. Cooke HA, Arndt J, Quan C, Shapiro RI, Wen D, Foley S, Vecchi MM, Preyer M. EFab domain substitution as a solution to the light-chain pairing problem of bispecific antibodies. MAbs 2018; 10:1248-59. Tiller KE, Li L, Kumar S, Julian MC, Garde S, Tessier PM. Arginine mutations in antibody complementarity-determining regions display context -dependent affinity/specificity trade-offs. J Biol Chem 2017; 292:16638-52. Dashivets T, Stracke J, Dengl S, Knaupp A, Pollmann J, Buchner J, Schlothauer T. Oxidation in the complementarity-determining regions differentially influences the properties of therapeutic antibodies. MAbs 2016; 8:1525-35. Lamberth K, Reedtz-Runge SL, Simon J, Klementyeva K, Pandey GS, Padkjaer SB, Pascal V, Leon IR, Gudme CN, Buus S, et al. Post hoc assessment of the immunogenicity of bioengineered factor VIIa demonstrates the use of preclinical tools. Sci Transl Med 2017; 9. Harding FA, Stickler MM, Razo J, DuBridge R. The immunogenicity of humanized and fully human antibodies. mAbs 2014; 2: 256-65. Sekiguchi N, Kubo C, Takahashi A, Muraoka K, Takeiri A, Ito S, Yano M, Mimoto F, Maeda A, Iwayanagi Y, et al. MHC-associated peptide proteomics enabling highly sensitive detection of immunogenic sequences for the development of therapeutic antibodies with low immunogenicity. MAbs 2018; 10:1168-81. Schachner L, Han G, Dillon M, Zhou J, McCarty L, Ellerman D, Yin Y, Spiess C, Lill JR, Carter PJ, et al . Characterization of chain pairing variants of bispecific IgG expressed in a single host cell by high-resolution native and denaturing mass spectrometry. Anal Chem 2016; 88:12122-7. Example 3 : Affinity maturation of modified antibodies produced in Example 2

The exemplary antibodies in Table I generated in Example 2 were subjected to affinity maturation to improve the affinity of the antibodies for their respective target antigens. Table I Antibody CDR L3* CDR H3* Exemplary candidates for affinity maturation ( up to about 20-40 fold to restore parental affinity ) Anti-HER2 T94D Parents The _KD of the modified antibody is approximately 20-fold lower than that of the unmodified parent** Parents W95D The _KD of the modified antibody is approximately 30-fold lower than that of the unmodified parent** anti-VEGFA V94D Parents The _KD of the modified antibody is comparable to that of the unmodified parent (and can be further affinity matured if necessary)** Parents Y95D The _KD of the modified antibody is approximately 38-fold lower than that of the unmodified parent** V94D:W96R Parents The _KD of the modified antibody is about 20 times lower than that of the unmodified parent** *Amino acid numbering is according to Kabat. ** See Table G3 .

Briefly, mutations were introduced into the CDRs of the antibodies in Table I to generate one or more polypeptide libraries (eg, phage-displayed or cell-surface-displayed libraries) for each antibody. One or more amino acid substitutions ( see Table 1 ) introduced into the CDR-L3 and/or CDR-H3 of each antibody to improve bispecific yield were kept fixed and not randomized during library construction. For example, as described in Wark et al. (2006) Adv Drug Deliv Rev. 58: 657-670; Rajpal et al. (2005) Proc Natl Acad Sci USA . 102: 8466-8471, followed by panning or cell Libraries are screened by sorting to identify antibody variants that bind the target antigen (ie, HER2, VEGFA, or VEGFC) with high affinity. Such variants are then isolated, and their affinity for their target antigen is determined, e.g., by surface plasmon resonance, and compared to the antibodies shown in Table I and the parental antibody from which the antibodies in Table I were derived. Comparison (see eg Table G3 ). Perform at least one round (such as at least any of 2, 3, 4, 5, 6, 7, 8, 9, or 10 rounds) of affinity maturation to identify high affinity anti-HER2 variants, high affinity anti-VEGFA variants, and high affinity Affinity-resistant VEGFC variants. Determining the sequence of antibody variants with high affinity for their respective target antigens.

Next, the variants identified in the screens described above were further analyzed to assess their impact on bispecific antibody yield. Briefly, high affinity anti-HER2, anti-VEGFA and anti-VEGFC variants were reprogrammed as bispecific antibodies. Exemplary bispecific antibodies include, but are not limited to, eg, anti-HER2/anti-CD3, anti-VEGFA/anti-ANG2, and anti-VEGFC/anti-CD3 (see Tables G1 and G2 above). For example, bispecific antibodies were expressed and purified according to the methods detailed in Example 1. As detailed in Example 1, the yield of correctly assembled bispecific antibodies is assessed, for example, by size exclusion chromatography, high resolution LCMS and/or SDS-PAGE gel analysis. Control experiments were performed in parallel using bispecific antibodies such as those shown in Tables G1 and G2 . Yields of bispecific antibodies comprising high affinity anti-HER2 antibody variants, high affinity anti-VEGFA variants or anti-VEGFC variants identified by library screening were compared with those comprising anti-HER2, anti-VEGFA or anti-VEGFC variants shown in Table 1 . The yields of bispecific antibodies against VEGFC antibodies were compared. Additional modified antibodies subjected to one or more affinity maturation steps and further assayed for improved affinity and BsAb yield (ie, as described above) are shown in Table G3 . Additional References Merchant et al. (2013) Proc Natl Acad Sci US A. 110(32): E2987-96 Julian et al. (2017) Scientific Reports. 7: 45259 Tiller et al. (2017) Front. Immunol. 8 : 986 Koenig et al. (2017) Proc Natl Acad Sci US A. 114(4): E486-E495 Yamashita et al. (2019) Structure. 27, 519-527 Payandeh et al. (2019) J Cell Biochem. 120 : 940-950 Richter et al. (2019) mAbs. 11(1): 166-177 Cisneros et al. (2019) Mol. Syst. Des. Eng. 4: 737-746

The foregoing examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and are within the scope of the appended claims.

Figures 1A and 1B provide high resolution liquid chromatography mass spectrometry (LCMS) data for an anti-LGR5/anti-IL4 bispecific antibody, ie, a representative example of low yield BsIgG. Figure 1A shows the mass envelopes for charge states 38+ and 39+. Figure 1B shows the corresponding deconvolved data. Figures 1C and ID provide a representative example of high resolution LCMS data for anti-SIRPα/anti-IL4 bispecific antibody, ie, intermediate yield BsIgG. Figure 1C shows the mass envelopes for charge states 38+ and 39+. Figure ID shows the corresponding deconvolved data. Figures IE and IF provide representative examples of high resolution LCMS data of anti-Met/anti-DR5 bispecific antibody, ie, high yield BsIgG. Figure IE shows the mass envelopes for charge states 38+ and 39+. Figure 1F shows the corresponding deconvolved data. Figure 2 provides the results of experiments performed to determine whether incorporation of _CH 1/ _CL charge pair substitution mutations increased BsIgG yield, demonstrating a strong intrinsic HC/LC pairing preference. Figure 3 illustrates the design of experiments performed to investigate the mechanistic basis for preferential HC/LC pairing in anti-EGFR/anti-MET BsIgG and anti-IL-4/anti-IL-13 BsIgG. The results of this experiment are provided in Table C. Figure 4A provides anti-MET antibody onatuzumab (onartuzumab) (see Merchant et al. (2013) PNAS USA 110: E2987-2996) (SEQ ID NO: 1) and anti-EGFR antibody D1.5 (see Schaefer et al. (2011) Cancer Cell 20: 472-486) (SEQ ID NO: 2) alignment of light chain variable domains (V _L ). Amino acid residues are numbered according to Kabat. CDRs from the sequence definition of Kabat et al. Sequences of Proteins of Immunological Interest. Bethesda, MD: NIH, 1991 and the structure definition of Chothia and Lesk (1987) J Mol Biol 196: 901-917 are shaded. Figure 4B provides an alignment of the heavy chain variable domains ( _VH ) of the anti-MET antibody onatuzumab (SEQ ID NO: 3) and the anti-EGFR antibody D1.5 (SEQ ID NO: 4). Amino acid residues are numbered according to Kabat. CDRs from the sequence definition of Kabat et al. Sequences of Proteins of Immunological Interest. Bethesda, MD: NIH, 1991 and the structure definition of Chothia and Lesk (1987) J Mol Biol 196: 901-917 are shaded. Figure 5A provides the results of experiments performed to assess the contribution of the complementarity determining region (CDR) L3 and CDR H3 of the anti-EGFR arm of the anti-EGFR/anti-MET bispecific antibody to the BsIgG yield. Also provided are the results of experiments performed to assess the contribution of CDR L3 and CDR H3 of the anti-MET arm of the anti-EGFR/anti-MET bispecific antibody to the BsIgG yield. Figure 5B provides the results of experiments performed to assess the contribution of CDR L3 and CDR H3 of the anti-IL-4 arm of an anti-IL-4/anti-IL-13 bispecific antibody to BsIgG yield. Also provided are the results of experiments performed to assess the contribution of CDR L3 and CDR H3 of the anti-IL-13 arm of the anti-IL-4/anti-IL-13 bispecific antibody to the BsIgG yield. Figure 6 provides a summary of experiments conducted to assess the contribution of CDR-L1 + CDR-H1, CDR-L2 + CDR-H2 and CDR-L3 + CDR-H3 of anti-EGFR/anti-MET bispecific antibodies to BsIgG yield result. Figure 7 provides the X-ray structure of the anti-MET Fab (PDB 4K3J) highlighting CDR L3 and CDR H3 contact residues. Figure 8A provides the anti-IL-13 antibody lebrikizumab (see Ultsch et al. (2013) J Mol Biol 425: 1330-1339) (SEQ ID NO: 5) and the anti-IL-4 antibody 19C11 (see Spiess Alignment of the light chain variable domains (V _L ) of et al. (2013) J Biol Chem 288: 265:83-93) (SEQ ID NO: 6). CDRs from Kabat's sequence definition and Chothia and Lesk's structure definition are shaded. Figure 8B provides an alignment of the heavy chain variable domains ( _VH ) of the anti-IL-13 antibody lenizumab (SEQ ID NO: 7) and the anti-IL-4 antibody 19C11 (SEQ ID NO: 8). Amino acid residues are numbered according to Kabat. CDRs from Kabat's sequence definition and Chothia and Lesk's structure definition are shaded. Figure 9 provides the X-ray structure of the anti-IL-13 Fab (PDB 4177) highlighting CDR L3 and CDR H3 contact residues. Figure 10A provides the results of experiments performed to assess the effect of: (a) CDR L3 and CDR H3 of the anti-CD3 arm of an anti-CD3/anti-HER2 bispecific antibody with the CDR of anti-MET Replace L3 and CDR H3; (b) replace CDR L3 and CDR H3 of the anti-HER2 arm of the anti-CD3/anti-HER2 bispecific antibody with CDR L3 and CDR H3 of anti-MET; (c) replace the anti-CD3/anti-HER2 bispecific antibody CDR L3 and CDR H3 of the anti-CD3 arm of the specific antibody were replaced with CDR L3 and CDR H3 of anti-IL-13; and (d) CDR L3 and CDR H3 of the anti-HER2 arm of the anti-CD3/anti-HER2 bispecific antibody were replaced Replaced with anti-IL-13 CDR L3 and CDR H3. Figure 10B provides the results of experiments performed to assess the effect of: (a) CDR L3 and CDR H3 of the anti-VEGFA arm of an anti-VEGFA/anti-ANG2 bispecific antibody with the CDR of anti-MET Replace L3 and CDR H3; (b) replace CDR L3 and CDR H3 of the anti-ANG2 arm of the anti-VEGFA/anti-ANG2 bispecific antibody with CDR L3 and CDR H3 of anti-MET; (c) replace the anti-VEGFA/anti-ANG2 bispecific antibody CDR L3 and CDR H3 of the anti-VEGFA arm of the specific antibody were replaced with CDR L3 and CDR H3 of anti-IL-13; and (d) CDR L3 and CDR H3 of the anti-ANG2 arm of the anti-VEGFA/anti-ANG2 bispecific antibody were replaced Replaced with anti-IL-13 CDR L3 and CDR H3. Figure 11 provides the results of experiments performed to assess the contribution of interchain disulfide bonds to BsIgG yield for the following bispecific antibodies: (1) anti-HER2/anti-CD3; (2) anti-VEGFA/anti-VEGFC; (3) ) anti-EGFR/anti-MET; and (4) anti-IL13/anti-IL-4.

Claims (12)

A method for improving the preferential pairing of a first heavy chain and a first light chain of a bispecific antibody comprising a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein the The first heavy chain and the first light chain form a pair (pair) that binds a first antigen, and wherein the second heavy chain and the second light chain form a pair that binds a second antigen, the method comprising the steps of: Substitution of the uncharged amino acid at position 94 of the light chain variable domain (V _L ) of the light chain with aspartic acid (D), the uncharged amine at position 96 of the V _L of the first light chain amino acid substitution with arginine (R), and the uncharged amino acid at position 95 of the heavy chain variable domain (V _H ) of the first heavy chain with aspartic acid (D), wherein Amino acid numbering is according to Kabat. The method of claim 1, wherein the substitutions at position 94 of the _VL , at position 96 of the _VL and at position 95 of the _VH are introduced into the anti-HER2 arm of the anti-HER2/anti-CD3 bispecific antibody, Introduced into the anti-VEGFA arm of the anti-VEGFA/anti-ANG2 bispecific antibody or into the anti-VEGFC arm of the anti-VEGFC/anti-CD3 bispecific antibody. The method of claim 1, further comprising subjecting the bispecific antibody to at least one affinity maturation step, wherein the substituted amino acid at position 94 of the _VL , the substituted amine at position 96 of the _VL The amino acid and the substituted amino acid at position 95 of the _VH were not randomized. The method according to claim 1, wherein the bispecific antibody is a human antibody, a humanized antibody or a chimeric bispecific antibody. The method according to claim 1, wherein the bispecific antibody comprises a human IgG Fc region. The method according to claim 5, wherein the human IgG Fc region is human IgG1, human IgG2, human IgG3 or human IgG4 Fc region. The method according to claim 6, wherein the human IgG Fc region comprises a first _CH 2 domain ( _CH 2 ₁ ), a first _CH 3 domain ( _CH 3 ₁ ), a second _CH 2 domain ( CH _{2 2} ) and a second _CH 3 domain; (a) wherein CH _{3 2} is altered such that within the _CH 3 ₁ /CH _{3 2} interface, one or more amino acid residues are modified with One or more amino acid residues of larger side chain volume are replaced, thereby generating _a protuberance ( _protuberance ) interacting with _CH31 on the surface of _CH32 ; and wherein _{CH31 is} changed to In the _CH 3 ₁ / _CH 3 ₂ interface, one or more amino acid residues are replaced by amino acid residues with smaller side chain volumes, resulting in the surface of _CH 3 ₁ and C _H 3 ₂ interaction pocket; or (b) wherein CH _{3 1} is altered such that within the _CH 3 ₁ /CH _{3 2} interface, one or more amino acid residues have larger side chains One or more amino acid residues in the volume are replaced, thereby creating a bump on the surface of CH _{3 1} that interacts with _CH 3 ₂ ; and wherein CH _{3 2} is changed so that in _CH 3 ₁ / In the _CH 3 ₂ interface, one or more amino acid residues are replaced by amino acid residues with smaller side chain volumes, thereby creating a concave interaction with CH _{3 1} on the surface of CH _{3 2} Cavity. The method according to claim 7, wherein the protrusion is a knob mutation. The method of claim 8, wherein the knob mutation comprises T366W, wherein the amino acid numbering is according to the EU index. The method according to claim 7, wherein the cavity is a hole mutation. The method according to claim 10, wherein the hole mutation comprises at least one, at least two or all three of T366S, L368A and Y407V, wherein the amino acid numbering is according to the EU index. An antibody produced by the method as claimed in claim 1.

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